Perhydrolase

ABSTRACT

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

The present application is a continuation application of U.S. application Ser. No. 10/581,014, filed Sep. 11, 2007, now U.S. Pat. No. 8,772,007, which is a U.S. National Stage application of International Application No. PCT/US04/40438, filed Dec. 3, 2004, which claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application Ser. No. 60/526,764, filed Dec. 3, 2003, which are hereby incorporated herein in their entireties.

FIELD OF THE INVENTION

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

BACKGROUND OF THE INVENTION

Detergent and other cleaning compositions typically include a complex combination of active ingredients. For example, most cleaning products include a surfactant system, enzymes for cleaning, bleaching agents, builders, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes. Despite the complexity of current detergents, there are many stains that are difficult to completely remove. Furthermore, there is often residue build-up, which results in discoloration (e.g., yellowing) and diminished aesthetics due to incomplete cleaning. These problems are compounded by the increased use of low (e.g., cold water) wash temperatures and shorter washing cycles. Moreover, many stains are composed of complex mixtures of fibrous material, mainly incorporating carbohydrates and carbohydrate derivatives, fiber, and cell wall components (e.g., plant material, wood, mud/clay based soil, and fruit). These stains present difficult challenges to the formulation and use of cleaning compositions.

In addition, colored garments tend to wear and show appearance losses. A portion of this color loss is due to abrasion in the laundering process, particularly in automated washing and drying machines. Moreover, tensile strength loss of fabric appears to be an unavoidable result of mechanical and chemical action due to use, wearing, and/or washing and drying. Thus, a means to efficiently and effectively wash colored garments so that these appearance losses are minimized is needed.

Cleaning compositions that comprise esterases, lipases and cutinases are well-known in the art. However, these enzymes have a very low ratio of perhydrolysis to hydrolysis. This results in the conversion of most of the ester substrate into acid, instead of the more desirable peracid. This is a serious drawback, since formula space and cost considerations render it feasible to include only a limited amount of substrate.

In sum, despite improvements in the capabilities of cleaning compositions, there remains a need in the art for detergents that remove stains, maintain fabric color and appearance, and prevent dye transfer. In addition, there remains a need for detergent and/or fabric care compositions that provide and/or restore tensile strength, as well as provide anti-wrinkle, anti-bobbling, and/or anti-shrinkage properties to fabrics, as well as provide static control, fabric softness, maintain the desired color appearance, and fabric anti-wear properties and benefits. In particular, there remains a need for the inclusion of compositions that are capable of removing the colored components of stains, which often remain attached to the fabric being laundered. In addition, there remains a need for improved methods and compositions suitable for textile bleaching.

In addition to the fabric and garment cleaning area, bleaching is commonly used in the pulp and paper industry. Prior to production of paper, pulp is typically treated to remove undesirable colored contaminants. This provides pulp that is suitable for production of paper of higher quality than pulp that is not treated to remove colored contaminants and other undesirable components present in pulp. For example, in the paper recycling industry, removal of ink is necessary. Although standard methods are suitable for deinking paper with oil or water-based inks, the increased use of electrostatic inks has made deinking problematic, as these inks are much more difficult to remove. There are various methods available for deinking paper, including the use of enzymes (See e.g., U.S. Pat. No. 5,370,770). However, there remains a need in the art for efficient, cost-effective methods for treatment of pulp for paper (recycled and new) product production.

Bleaching is also commonly used in the personal care market (e.g., dental whiteners, hair bleachers, etc.). Although personal care bleaching products have improved over the years, there remains a need for mild, easy to use, cost-effective bleaching methods for this setting.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

In some embodiments, the present invention provides compositions comprising at least one perhydrolase, wherein the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1.

The present invention also provides isolated perhydrolases, wherein the perhydrolases exhibit a perhydrolysis to hydrolysis ratio that is greater than 1. In some preferred embodiments, the perhydrolase is M. smegmatis perhydrolase. In alternative preferred embodiments, the perhydrolase is at least approximately about 35% homologous to M. smegmatis perhydrolase. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2. In some preferred embodiments, the perhydrolases have immunological cross-reactivity with M. smegmatis perhydrolase. In still further embodiments, the perhydrolase is at least a portion of M. smegmatis perhydrolase, wherein the perhydrolase has a perhydrolysis to hydrolysis ration that is greater than 1. In alternative embodiments, the perhydrolase is a structural homologue of M. smegmatis perhydrolase, in which the active site is homologous to at least one amino acid selected from the group consisting of S11, D192, and H195 of the M. smegmatis perhydrolase.

The present invention also provides isolated perhydrolase variants having amino acid sequences comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, at least one modification is made at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the modified amino acid is selected from the group consisting of Cys7, Asp10, Ser11, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194, and Phe196. In further embodiments, the modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of M1, K3, R4, I5, L6, C7, D10, S11, L12, T13, W14, W16, G15, V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31, V32, R33, W34, T35, G36, L38, Q40, Q41, D45, L42, G43, A44, F46, E47, V48, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, I60, D61, D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77, L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97, Y99F100, R101, R102, P104, L105, D106, I107, A108, L109, G110, M111, S112, V113, L114, V115, T116, Q117, V118, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P146, P148, W149, F150, I153, F154, I194, and F196.

In some preferred embodiments, the variant perhydrolase exhibits a change in peracid hydrolysis compared to the wild-type perhydrolase. In some embodiments, the change in peracid hydrolysis is a decrease, while in other embodiments, the change in peracid hydrolysis is an increase.

In some alternative preferred embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.1 or less, in comparison with wild-type perhydrolase. In alternative preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, L12, G15, P18, R27, W34L38, A44, E51, G52, L53, S54, T58, R67, L68, S72, A79, T80, D85, L86, V87, N94, K97, R101, V118, L119, G124, G126, and I194.

In further alternative embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.2 or less, in comparison with wild-type perhydrolase. In yet additional embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, I5, D10, L12, W14, G15, P18, V19, T25, R27, W34, L38, A44, I49, E50, E51, G52, L53, S54, A55, R56, T58, N59, D62, T64, D65, R67, L68, N69, S72, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, R101, L82, P83, L86, V87, N94, T96, K97, F100, R101, L109, M111, L114, V118, L119, A122, G124, G126, T127, Y129, W149, and I194.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.3 or less, in comparison with wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, I5, D10, L12, W14, G15, L12, P18, V19, G22, A23, T25, E26, R27, W34, G36, L38, Q41, L42, G43, A44, I49, E50, E51, G52, L53, S54, A55, R56, T57, N59, T58, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, Y99, F100, R101, R102, P104, L109, G110, M111, L114, V118, L119, A122, G124, V125, G126, T127, Y129, W149, F154, and I194.

In yet further embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.4 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, I5, L6, D10, S11, L12, W14, G15, W16, P18, V19, G22, A23, T25, E26, R27, F28, W34, T35, G36, L38, Q41, L42, G43, A44, D45, E47, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, T58, I60, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, P66, T96, K97, Y99, F100, R101, R102, P104, I107, L109, G110, M111, S112, L114, V118, L119, S121, A122, G124, V125, G126, T127, Y129, W149, F150, F154, I194, and F196.

In some embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.5 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54, S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.6 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54, S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, V125, V19, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In yet additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.7 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54, S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84, L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In still further embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.8 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y1d29, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54, S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84, L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52, I107, I194, I49, I5, I60, I89, L114, L42, L53, L68, L78, L84, M111, N59, N94, P146, P24, P30, P63, P66, P83, Q117, R101, R4, S112, S121, S72, T116, T120, T127, T13, T57, T96, V113, V125, V17, V19, V32, V87, W149, Y129, Y73, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 1.5 or greater, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101, R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38, L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118, V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119, L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54, S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84, L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52, I107, I194, I49, I5, I60, I89, L114, L42, L53, L68, L78, L84, M111, N59, N94, P146, P24, P30, P63, P66, P83, Q117, R101, R4, S112, S121, S72, T116, T120, T127, T13, T57, T96, V113, V125, V17, V19, V32, V87, W149, Y129, and Y73, Y99, A108, A44, C7, D10, D106, D31, D61, D85, E26, E51, F100, F28, F46, G110, G22, G36, G43, G52, G70, I107, I153, I49, I5, I89, K3, L105, L53, L6, L78, L86, M1, N69, P104, P146, P18, P24, P30, P83, Q117, Q40, Q41, R102, R27, R33, R4, S121, S72, S76, T120, T128, T13, T35, T80, T96, V115, V118, V32V48, V87, W34, G190, V191, G193, T197, E198, A199, R202, D203, G205, V206, A209, E210, Q211, S214, and L215.

In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis between about 1.2 and about 1.5, in comparison with wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, C7, D106, D31, D61, D85, E26, E50, E51, F100, F150, F28, F46, G110, G126, G22, G70, I107, K3, L105, L42, L6, L78, M111, N59, N69, P104, P146, P148, P18, P30, P63, Q117, Q40, Q41, R102, R27, R33, R4, S54, S76, T116, T120, T128, T64, T80, T96, V113, V115, V118, W34, and Y73.

In yet further embodiments, the present invention provides variant perhydrolases in which the variant perhydrolases exhibit a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is at least about 1.2. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, I49, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, R101, T103, P104, L105, D106, I107, L109, M111, V113, Q117, V118, S121, G124, V125, G126, T127, P148, F150, I153, F154, and F196.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.8 or less. In some embodiments, the variant perhydrolase comprising at least one modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D45, D61, D62, D65, D85, E26, E47, E50, E51, F100, F150, F154, F196, F28, F46, G110, G124, G126, G15, G22, G36, G52, G70, I107, I153, I194, I49, I5, I60, I89, K3, K97, L105, L109, L114, L119, L12, L38, L42, L53, L6, L68, L78, L82, L84, K86, M1, M111, N59N94, P146, P18, P24, P30, P66, P83, Q40, Q41, R101, R102, R27, R33, R4, R56, R67, S11, S112, S54, S72, S76, T103, T116, T120, T127, T128, T13, T25, T35, T57, T64, T80, T96, V113, V115, V118, V125, V17, V19, V32, V48, V87, W13, W149, W16, W34, Y129, Y73, and Y99.

In alternative embodiments, the present invention provides variant perhydrolases comprising at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D31, D45, D61, D62, D65, D85, E26, E47, E50, E51, F100, F150, F154F196, F28, F46, G110, G124, G126, G15, G22, G36, G43, G52, G70, I107, I153, I194, I49, I5, I60, I89, K3, K97, L105, L109, L114, L119, L12, L38, L42, L53, L6, L68, L78, L82, L84, L86, M1, M111, N59, N69, N94, P104, P146, P148, P18, P24, P30, P63, P66, P83, Q117, Q40, Q41, R101, R102, R27, R33, R4, R56, R67, S11, S112, S121, S54, S72, S76, T103, T116, T120, T127, T128, T13, T25, T35, T57, T58, T64, T80, T96, V113, V115, V118, V125, V17, V19, V32, V48, V87, W14, W149, W16, W34, Y129, Y73, and Y99.

In yet additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 1.2 and about 2. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, I49, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, R101, T103, P104, L105, D106, I107, L109, M111, V113, Q117, V118, S121, G124, V125, G126, T127, P148, F150, I153, F154, F196, G190, E198, A199, R202, D203, V206, A209, E210, Q211, and V212.

In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2 and about 2.5. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A44, C7, D10, D85, D95, E26, E47, I107, L12, L42, P104, P148, S54, Q40, Q117, D203, V206, E210. In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2.5 and about 3. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A44, C7, I107, K97, L12, L78, P104, Q40, and V125.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 3.0 and about 5. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of D10, D85, L53, L78, and S54.

In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.1 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, and W34. In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.2 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, and Y73.

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.3 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, and Y129.

In yet additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.4 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, and V87.

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.5 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, and Y129.

In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.6 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising t least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, g126, G36, G52, I107, I5, I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115, V118, V32, V48, V87, W149, and Y73.

In yet further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.7 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, g126, G36, G52, I107, I5, I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115, V118, V32, V48, V87, W149, Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, F100, F28, F46, G110, G126, G52, G70, I107, I49, I5, I60, I89, L109, L114, L12, L38, L68, L82, L86, M111, N59, N94, P83, R102, R33, R4, S112, S72, S76, T103, T116, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, and Y99.

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.8 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25, T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97, L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4, R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119, L12, L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27, R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, g126, G36, G52, I107, I5, I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115, V118, V32, V48, V87, W149, Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, F100, F28, F46, G110, G126, G52, G70, I107, I49, I5, I60, I89, L109, L114, L12, L38, L68, L82, L86, M111, N59, N94, P83, R102, R33, R4, S112, S72, S76, T103, T116, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, Y99, A108, A122, A29, A55, C77, D10, D106, D45, D61, D62, D65, D85, E47, E50, F100, F150, F28, F46, G110, G124, G126, G15, G36, I153, I194, I5, I60, I89, K3, K97, L105, L109, L114, L119, L38, L42, L68, L84, L86, M1, N59, P24, P30, P83, R101, R27, R4, R56, S112, S54, S76, T103, T116, T120, T127, T128, T13, T35, T64, V113, V17, V19, V32, V48, V87, Y129, Y73, and Y99.

The present invention also provides perhydrolase variants, wherein the perhydrolase variants exhibit greater perhydrolysis activity and decreased peracid hydrolysis activity as compared to wild-type perhydrolase. In some embodiments, the variant perhydrolases exhibit perhydrolysis activity ratio of at least about 1.2, and peracid hydrolysis activity ratio of about 0.8 or less, as compared to wild-type perhydrolase. In alternative embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A29, A44, A55, A71, A79, C7, D10, D106, D31, D85, E26, E47, F150, F154, F196, F28, G124, G126, G36, G43, I153, L109, L42, L53, L109, L42, L53, L109, L42, L53, L68, L82, L86, M111, N69, P104, P148, P18, P63, P66, P83, Q117, Q40, R101, R67, S54, S121, S72, S76, T25, T64, V115, and V19.

In additional embodiments, the perhydrolase exhibits perhydrolysis activity ratio of at least about 1.2, a peracid hydrolysis activity ratio of about 0.8 or less, and a protein concentration ratio of at least 0.5, as compared to wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A29, A44, A71, A79, C7, D85, E26, E47, E51, F150, F154, F196, F28, G124, G126, G36, I153, L109, L12, L53, L68, L82, M111, N69, P104, P148, P18, P63, P66, P83, Q117, Q40, R101, R67, S121, S54, S72, S76, T25, T64, V125, and V19.

The present invention provides variant perhydrolases that exhibit an increase in expression of the perhydrolase variants, as compared to the expression of wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A2, I5, C7, F8, S11, L12, T13, W14, W16, V17, P18, V19, E20, G22, A23, P24, T25, A29, P30, V32, T35, G36, V37, A39, F46, E47, S54, A55, R56, T58, I60, D61, D62, P63, T64, P66, R67, L68, N69, G70, S72, Y73, L74, P75, S76, C77, L78, A79, T80, L82, P83, L84, L86, I89, T93, T96, K97, A98, Y99, F100, R101, R102, T103, P104, L105, D106, I107, A108, L109, G110, 5112, V113, L114, V115, T116, Q117, V118, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P130, P132, K133, L135, V136, 5138, P141, L142, A143, M145, H147, W149, F150, Q151, I153, G157, Q159, T161, T162, L164, A165, R166, V167, Y168, A170, L171, A172, M175, K176, P178, A182, G183, S184, V185, I186, T188, 1194, F196, V191, N201, L208, A209, Q211, Q213, S214, L215, and L216.

The present invention also provides isolated proteins comprising homologs of M. smegmatis perhydrolase, wherein the homologs are proteins within the SGNH-hydrolase family of proteins. In alternative preferred embodiments, the isolated proteins have at least about 35% identity with the amino acid sequence of M. smegmatis perhydrolase, in which the protein comprises at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, S11, D192, and H195. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2.

The present invention also provides isolated proteins having at least about 38% identity with the amino acid sequence of M. smegmatis perhydrolase, wherein the protein exhibits perhydrolysis activity. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2.

The present invention also provides homologs of M. smegmatis perhydrolase, wherein the homologs are perhydrolases comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In preferred embodiments, the homologs exhibit perhydrolysis. In some particularly preferred embodiments, the homologs exhibit a perhydrolysis to hydrolysis ratio that is great than about 1. In still further embodiments, the homologs are immunologically cross-reactive with antibodies raised against M. smegmatis perhydrolase. In yet additional embodiments, antibodies raised against the homolog cross-react with M. smegmatis perhydrolase.

The present invention also provides isolated proteins having at least about 35% identity with the amino acid sequence of at least one M. smegmatis perhydrolase homolog, wherein the proteins exhibit perhydrolysis activity.

In some particularly preferred embodiments, the present invention provides proteins having perhydrolase activity, wherein the proteins are in the form of a multimer in solution. In some more preferred embodiments, the protein is a perhydrolase that comprises a dimer. In alternative particularly preferred embodiments, the protein is a perhydrolase that comprises an octamer. In still further embodiments, the protein is in the form of a multimer in solution and the protein is selected from the group consisting of M. smegmatis perhydrolase, M. smegmatis perhydrolase homologs, and M. smegmatis perhydrolase variants. In yet further embodiments, the protein is selected from the group consisting of modified serine hydrolases and modified cysteine hydrolases, wherein the modified serine hydrolases or modified cysteine hydrolases comprise increased perhydrolase activity as compared to unmodified serine hydrolases or unmodified cysteine hydrolases

The present invention also provides proteins having perhydrolase activity, wherein the protein comprises at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In some embodiments, the protein is obtained from a member of the Rhizobiales. In some preferred embodiments, the protein is obtained from a member of the genus Mycobacterium.

The present invention also provides isolated genes identified using at least one primer selected from the group consisting of SEQ ID NOS:21-69.

The present invention also provides methods for identifying a perhydrolase, comprising the steps of: identifying source of the perhydrolase; analyzing the source to identify sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT; expressing the sequences identified in step b) to produce the perhydrolase; and testing the perhydrolase for perhydrolysis activity.

In some embodiments, the analyzing step is an amplification step wherein the primer sequences set forth in SEQ ID NOS:21-69 are used to amplifying the sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In still further embodiments, the source is selected from the group consisting of environmental sources and metagenomic sources. The present invention also provides proteins identified using the methods set forth herein. The present invention further provides isolated nucleic acid sequences encoding the proteins identified using the methods set forth herein. In some particularly preferred embodiments, the proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than about 1. In still further embodiments, the proteins exhibit a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase. In yet additional embodiments, the proteins comprise at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, S11, D192, and H195.

In further embodiments, the analyzing step comprises searching at least one amino acid database. In yet further embodiments, the analyzing step comprises searching at least one nucleic acid database to identify nucleic acid sequences encoding the amino acid sequences of the perhydrolase. In still further embodiments, the source is selected from the group consisting of environmental sources and metagenomic sources. The present invention further provides isolated nucleic acid sequences encoding the proteins identified using the methods set forth herein. In some particularly preferred embodiments, the proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than about 1. In still further embodiments, the proteins exhibit a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase. In yet additional embodiments, the proteins comprise at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, S11, D192, and H195, as set forth in SEQ ID NO:2.

The present invention also provides variant perhydrolases having altered substrate specificities as compared to wild-type M. smegmatis perhydrolase. In some embodiments, the variant perhydrolases have altered para nitrophenyl caproate (PNC) activity, as compared to wild-type M. smegmatis perhydrolase.

The present invention also provides variant perhydrolases having altered pI values as compared to wild-type M. smegmatis perhydrolase. In some embodiments, the variant perhydrolases comprise at least one positively charged mutation, while in alternative embodiments, the variant perhydrolases comprise at least one negatively charged mutation.

The present invention also provides variant perhydrolases that have increased stability, as compared to wild-type M. smegmatis perhydrolase. In some preferred embodiments, the stability of the variant perhydrolase is selected from the group consisting of thermostability, enzymatic stability, and chemical stability.

The present invention also provides variant perhydrolases, wherein the variant perhydrolase exhibits at least one altered surface property. In some preferred embodiments, the variants comprise at least one mutation comprising at least one substitution at sites selected from the group consisting of the residues set forth in Table 15-1

The present invention also provides perhydrolase variants having at least one improved property as compared to wild-type perhydrolase.

The present invention also provides expression vectors comprising a polynucleotide sequence encoding at least one perhydrolase variant. The present invention further provides host cells comprising at least one such expression vector. In some preferred embodiments, a host cell is selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by the host cells.

The present invention also provides compositions comprising at least a portion of at least one perhydrolase. In some preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2. In further embodiments, the perhydrolase is encoded by a polynucleotide sequence comprises SEQ ID NO:1. In additional embodiments, the sequence comprises at least a portion of SEQ ID NO:1. In further embodiments, the present invention provides expression vectors comprising the polynucleotide sequence encoding at least a portion of at least one perhydrolase. The present invention also provides host comprising at least one expression vectors. In some embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by these host cells.

The present invention also provides variant perhydrolases, wherein the perhydrolases comprise at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein the variant perhydrolase has better performance in at least one property, compared to wild-type M. smegmatis perhydrolase.

The present invention further provides isolated polynucleotides comprising a nucleotide sequence (i) having at least about 70% identity to SEQ ID NO:1, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ID NO:1, under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the present invention also provides vectors comprising these polynucleotide sequences. In additional embodiments, the present invention also provides host comprising at least one expression vectors. In some embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by these host cells.

The present invention also provides polynucleotides comprising a sequence complementary to at least a portion of the sequence set forth in SEQ ID NO:1.

The present invention also provides methods of producing enzymes having perhydrolase activity, comprising: transforming a host cell with an expression vector comprising a polynucleotide having at least 70% sequence identity to SEQ ID NO:1; cultivating the transformed host cell under conditions suitable for the host cell to produce the perhydrolase; and recovering the perhydrolase. In some preferred embodiments, the host cell is selected from the group consisting of Streptomyces, Pantoea, Escherichia, and Bacillus species.

The present invention also provides probes comprising a 4 to 150 polynucleotide sequence substantially identical to a corresponding fragment of SEQ ID NO:1, wherein the probe is used to detect a nucleic acid sequence coding for an enzyme having perhydrolase activity.

The present invention also provides cleaning compositions comprising: a) at least 0.0001 weight percent of a perhydrolase that exhibits a perhydrolysis to hydrolysis ratio that is greater than 1; b) a molecule comprising an ester moiety; and c) optionally, an adjunct ingredient.

The present invention further provides cleaning compositions comprising: a) at least 0.0001 weight percent of a perhydrolase that exhibits a perhydrolysis to hydrolysis ratio that is greater than 1; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, the peroxygen source being selected from the group consisting of: a per-salt; an organic peroxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture, and mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient.

The present invention also provides cleaning compositions comprising: a) from about 0.0001 to about 1 weight percent of a variant perhydrolase having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, the peroxygen source being selected from the group consisting of: a per-salt; an organic peroxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture; and mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient. In some preferred embodiments, the cleaning compositions further comprise at least one adjunct ingredient. In some particularly preferred embodiments, the adjunct ingredient is selected from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removalanti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof.

In additional embodiments, the present invention provides cleaning compositions wherein: the perhydrolase exhibits a perhydrolysis to hydrolysis molar ratio that is greater than about 0.1; the per-salt is selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof; the carbohydrate is selected from the group consisting of mono-carbohydrates, di-carbohydrates, tri-carbohydrates, oligo-carbohydrates and mixtures thereof; the carbohydrate oxidase is selected from the group consisting of aldose oxidase (IUPAC classification EC1.1.3.9), galactose oxidase (IUPAC classification EC1.1.3.9), cellobiose oxidase (IUPAC classification EC1.1.3.25), pyranose oxidase (IUPAC classification EC1.1.3.10), sorbose oxidase (IUPAC classification EC1.1.3.11) hexose oxidase (IUPAC classification EC1.1.3.5). glucose oxidase (IUPAC classification EC1.1.3.4) and mixtures thereof; and the molecule comprising an ester moiety has the formula:

R¹O_(x)[(R²)_(m)(R³)_(n)]p

(i) wherein R¹ is a moiety selected from the group consisting of H, substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl;

(ii) each R² is an alkoxylate moiety;

(iii) R³ is an ester-forming moiety having the formula:

R⁴CO— wherein R⁴ is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl;

(iv) x is 1 when R¹ is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R¹;

(v) p is an integer that is equal to or less than x;

(vi) m is an integer from 0 to 50; and

(vii) n is at least 1

In alternative embodiments, the present invention provides cleaning compositions wherein: a) R¹ is an C₂-C₃₂ substituted or unsubstituted alkyl or heteroalkyl moiety; b) each R² is independently an ethoxylate or propoxylate moiety; and c) m is an integer from 1 to 12. In some embodiments, R³ is an ester-forming moiety having the formula: R⁴CO— wherein R⁴ is: a) a substituted or unsubstituted alkyl, alkenyl or alkynyl moiety comprising from 1 to 22 carbon atoms; or b) a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety comprising from 4 to 22 carbon atoms.

In still further embodiments of the cleaning compositions, the molecule comprising the ester moiety has the formula:

R¹O_(x)[(R²)_(m)(R³)_(n)]_(P)

wherein: a) R¹ is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, the R¹ moiety that comprises an amine moiety being selected from the group consisting of substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; b) each R² is an alkoxylate moiety; c) R³ is an ester-forming moiety having the formula: R⁴CO— wherein R⁴ may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; d) x is 1 when R¹ is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R′; e) p is an integer that is equal to or less than x; f) m is an integer from 0 to 12; and g) n is at least 1.

In still further embodiments of the present cleaning compositions, the molecule comprising an ester moiety has a weight average molecular weight of less than 600,000 Daltons. In yet additional embodiments, an adjunct ingredient is selected from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removalanti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof.

The present invention further provides methods of cleaning comprising the steps of: a) contacting a surface and/or an article comprising a fabric with any of the cleaning compositions provided above and/or a composition comprising any of the cleaning compositions provided above; and b) optionally washing and/or rinsing the surface or material.

In alternative embodiments, the present invention provides methods of cleaning, the method comprising the steps of: a) contacting a surface and/or an article comprising a fabric with any suitable cleaning composition provided above and/or a composition comprising any suitable cleaning provided above; and b) optionally washing and/or rinsing the surface or material.

The present invention also provides bleaching compositions comprising at least one perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase variant having at least one improved property as compared to wild-type perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase variant comprising at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein the variant perhydrolase has better performance in at least one property compared to wild-type M. smegmatis perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising at least one perhydrolase that is at least approximately about 35% homologous to M. smegmatis perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant having at least one improved property as compared to wild-type perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant comprising at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein the variant perhydrolase has better performance in at least one property compared to wild-type M. smegmatis perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprising at least one perhydrolase that is at least approximately about 35% homologous to M. smegmatis perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

In some preferred embodiments, the perhydrolase is at least approximately 70% homologous to M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the present invention provides perhydrolases that cross react with antibody generated against M. smegmatis perhydrolase, particularly that comprising the amino acid sequence set forth in SEQ ID NO:2. In further embodiments, the present invention provides perhydrolases that are structural homologs of the M. smegmatis perhydrolase, in which active site comprises sites homologous to S11, D192, and H195 of the M. smegmatis perhydrolase. In yet additional embodiments, the present invention provides perhydrolases comprising one or more modifications at the following residues: Cys7, Asp10, Ser11, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194, and Phe196. However, it is not intended that the present invention be limited to perhydrolases with these modifications only at these residues, as perhydrolases with other modifications also find use with the present invention.

In some embodiments, at least one perhydrolase of the present invention is used in a cleaning process wherein an article to be cleaned is exposed to a sufficient amount of the at least one perhydrolase under conditions such that the perhydrolase cleans and/or bleaches, and/or decolorizes any/all stains present on the article (e.g., laundry and dish detergents). In some embodiments, the cleaning further comprises disinfecting. In some embodiments, the article cleaned, bleached and/or disinfected using at least one perhydrolase of the present invention comprises textiles and/or hard surfaces, while in other embodiments, the article is paper or pulp, and in still further embodiments, at least one perhydrolase is used as a personal care product to whiten or bleach hair, teeth, skin, etc. Thus, in some embodiments, the present invention provides compositions for use in various cleaning, bleaching, and/or disinfecting applications. Indeed, it is not intended that the present invention be limited to any particular application.

In some preferred embodiments, the perhydrolase comprises SEQ ID NO:2. In some preferred alternative embodiments, the perhydrolase is encoded by the nucleic acid sequence set forth in SEQ ID NO:1.

In some embodiments, the present invention provides enzymes with activities that result in high peracidacid ratios. In alternative embodiments, the present invention provides the perhydrolase of Mycobacterium smegmatis, as well as sequence and/or structural homologs of this protein. In additional embodiments, the present invention provides enzymes that have been modified so as to express perhydrolase activity with a high perhydrolysis to hydrolase ratio either in addition to or instead of the enzyme's original activity. In additional embodiments, the present invention provides modified enzymes with altered substrate specificity, Km, kcat, perhydrolase activity, and/or peracid degradation activity.

In additional embodiments, the present invention provides means to identify, produce, and characterize enzymes that comprise the perhydrolysis activity of the present invention. The present invention further provides methods and compositions comprising at least one perhydrolase for cleaning, disinfecting, bleaching, and other applications, including but not limited to paper and pulp bleaching, fabric and garment cleaning, hard surface cleaning, and personal care applications (e.g., oral care, hair care, and skin care). In some preferred embodiments, the present invention provides methods and compositions for bleaching cotton and other fabrics. Indeed, the present invention finds use in the bleaching and cleaning of various textiles. It is not intended that the present invention be limited to any particular setting, application or use, as it is contemplated that it will find use in numerous areas where an enzymatic generation of peracids is desired over the use of preformed peracids or hydrogen peroxide or other bleaching chemicals, under conditions including but not limited to a wide range of pHs and temperatures. The present invention also finds use in applications where peracid hydrolysis is useful, such as in the clean up of peracids.

Furthermore, the present invention provides means to produce perhydrolase enzymes suitable for cleaning, disinfecting, bleaching, and other applications, including personal care.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide a phylogenetic tree of M. smegmatis perhydrolase and other related sequences.

FIG. 2 provides an overview phylogenetic tree, showing the major branches of the bacteria and the origin of the active clonessequences compared to M. smegmatis.

FIG. 3 provides a schematic of four structural families of serine hydrolases, including perhydrolase (SGNH-hydrolase family), chymotrypsin, subtilisin, and α/β hydrolase.

FIG. 4 provides a diagram of the structure of the perhydrolase fold.

FIG. 5 provides a map of plasmid pET26-M4aE11.

FIG. 6 provides a purification table showing the enzyme activity of the enzyme of the present invention through various steps in the purification process.

FIG. 7 provides a graph which shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 40 minutes.

FIG. 8 provides a graph showing the peracid production by 30 mM acetate equivalents and 29 mM hydrogen peroxide, tested at various pHs. These results show that using the perhydrolase composition of the present invention, there is peracid generation over a wide pH range. In contrast, with TAED and hydrogen peroxide, peracid generation is limited to alkaline conditions.

FIG. 9 provides a graph showing the peracid production by 0.1 ppm perhydrolase enzyme in 30 mM ethyl acetate and 20 mM hydrogen peroxide at various temperatures. These results show that the perhydrolase of the present invention works at a wide range of temperatures, including low temperatures.

FIG. 10 provides a graph showing the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 4, 10, and 30 minutes.

FIG. 11 provides a graph showing the ratio of peracetic acid to acetic acid generated by various enzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10 minutes.

FIG. 12 provides a map of plasmid pMSATNcoI.

FIG. 13 provides a map of plasmid pMSATNcol-1.

FIG. 14 provides a map of plasmid pAH505.

FIG. 15 provides a map of plasmid pSFNASally.

FIG. 16 provides a map of plasmid pCP606.

FIG. 17 provides a map of plasmid pCP649.

FIG. 18 provides a map of plasmid pSECGT-MSAT.

FIG. 19 provides a map of plasmid pSEGT-phdA4.

FIG. 20 provides a map of plasmid pMC355rbs.

FIG. 21 provides a graph showing the degree of bleaching by three detergents tested alone and in comparison with the M. smegmatis perhydrolase of the present invention.

FIG. 22 provides a graph showing the bleaching ability of the M. smegmatis perhydrolase tested on cotton.

FIG. 23 provides a graph showing the bleaching ability of the M. smegmatis perhydrolase tested on linen.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. In particular, the present invention provides improved methods and compositions comprising perhydrolysis enzymes with high peracidacid ratios for cleaning, bleaching, disinfecting and other applications. In some preferred embodiments, the present invention provides improved methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA fields, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition (Cold Spring Harbor), [1989]); and Ausubel et al., “Current Protocols in Molecular Biology” [1987]). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms “a”, “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein, the term “bleaching” refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and under appropriate pH and temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include but are not limited to ClO₂, H₂O₂, peracids, NO₂, etc.

As used herein, the term “disinfecting” refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present invention be limited to any particular surface, item, or contaminant(s) or microbes to be removed.

As used herein, the term “perhydrolase” refers to an enzyme that is capable of catalyzing a reaction that results in the formation of sufficiently high amounts of peracid suitable for applications such as cleaning, bleaching, and disinfecting. In particularly preferred embodiments, the perhydrolase enzymes of the present invention produce very high perhydrolysis to hydrolysis ratios. The high perhydrolysis to hydrolysis ratios of these distinct enzymes makes these enzymes suitable for use in a very wide variety of applications. In additional preferred embodiments, the perhydrolases of the present invention are characterized by having distinct tertiary structure and primary sequence. In particularly preferred embodiments, the perhydrolases of the present invention comprises distinct primary and tertiary structures. In some particularly preferred embodiments, the perhydrolases of the present invention comprise distinct quaternary structure. In some preferred embodiments, the perhydrolase of the present invention is the M. smegmatis perhydrolase, while in alternative embodiments, the perhydrolase is a variant of this perhydrolase, while in still further embodiments, the perhydrolase is a homolog of this perhydrolase. In further preferred embodiments, a monomeric hydrolase is engineered to produce a multimeric enzyme that has better perhydrolase activity than the monomer. However, it is not intended that the present invention be limited to this specific M. smegmatis perhydrolase, specific variants of this perhydrolase, nor specific homologs of this perhydrolase.

As used herein, the term “multimer” refers to two or more proteins or peptides that are covalently or non-covalently associated and exist as a complex in solution. A “dimer” is a multimer that contains two proteins or peptides; a “trimer” contains three proteins or peptides, etc. As used herein, “octamer” refers to a multimer of eight proteins or peptides.

As used herein, the phrase “perhydrolysis to hydrolysis ratio” is the ratio of the amount of enzymatically produced peracid to that of enzymatically produced acid by the perhydrolase, under defined conditions and within a defined time. In some preferred embodiments, the assays provided herein are used to determine the amounts of peracid and acid produced by the enzyme.

As used herein, “personal care products” means products used in the cleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth, including, but not limited to shampoos, body lotions, shower gels, topical moisturizers, toothpaste, and/or other topical cleansers. In some particularly preferred embodiments, these products are utilized on humans, while in other embodiments, these products find use with non-human animals (e.g., in veterinary applications).

As used herein, “pharmaceutically-acceptable” means that drugs, medicaments and/or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and other animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefitrisk ratio.

As used herein, “cleaning compositions” and “cleaning formulations” refer to compositions that find use in the removal of undesired compounds from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes) etc. The term encompasses any materialscompounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the perhydrolase and other enzyme(s) used in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.

The terms further refer to any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).

Indeed, the term “cleaning composition” as used herein, includes unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types.

As used herein, the terms “detergent composition” and “detergent formulation” are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In some preferred embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., “laundry detergents”). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to perhydrolase, the term encompasses detergents that contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.

As used herein, “enhanced performance” in a detergent is defined as increasing cleaning of bleach-sensitive stains (e.g., grass, tea, wine, blood, dingy, etc.), as determined by usual evaluation after a standard wash cycle. In particular embodiments, the perhydrolase of the present invention provides enhanced performance in the oxidation and removal of colored stains and soils. In further embodiments, the perhydrolase of the present invention provides enhanced performance in the removal and/or decolorization of stains. In yet additional embodiments, the perhydrolase of the present invention provides enhanced performance in the removal of lipid-based stains and soils. In still further embodiments, the perhydrolase of the present invention provides enhanced performance in removing soils and stains from dishes and other items.

As used herein the term “hard surface cleaning composition,” refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like. Such compositions are provided in any form, including but not limited to solids, liquids, emulsions, etc.

As used herein, “dishwashing composition” refers to all forms for compositions for cleaning dishes, including but not limited to granular and liquid forms.

As used herein, “fabric cleaning composition” refers to all forms of detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms.

As used herein, “textile” refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics. The term encompasses yarns made from natural, as well as synthetic (e.g., manufactured) fibers.

As used herein, “textile materials” is a general term for fibers, yarn intermediates, yarn, fabrics, and products made from fabrics (e.g., garments and other articles).

As used herein, “fabric” encompasses any textile material. Thus, it is intended that the term encompass garments, as well as fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material.

As used herein, the term “compatible,” means that the cleaning composition materials do not reduce the enzymatic activity of the perhydrolase to such an extent that the perhydrolase is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.

As used herein, “effective amount of perhydrolase enzyme” refers to the quantity of perhydrolase enzyme necessary to achieve the enzymatic activity required in the specific application (e.g., personal care product, cleaning composition, etc.). Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like.

As used herein, “non-fabric cleaning compositions” encompass hard surface cleaning compositions, dishwashing compositions, personal care cleaning compositions (e.g., oral cleaning compositions, denture cleaning compositions, personal cleansing compositions, etc.), and compositions suitable for use in the pulp and paper industry.

As used herein, “oral cleaning compositions” refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Oral care compositions that find use in conjunction with the perhydrolases of the present invention are well known in the art (See e.g., U.S. Pat. Nos. 5,601,750, 6,379,653, and 5,989,526, all of which are incorporated herein by reference).

As used herein, “pulp treatment compositions” refers to the use of the present perhydrolase enzymes in compositions suitable for use in papermaking. It is intended that the term encompass compositions suitable for the treatment of any pulp material, including wood, as well as non-wood materials, such as “agricultural residues” and “fiber crops,” including but not limited to wheat straw, rice straw, corn stalks, bagasse (sugar cane), rye grass straw, seed flax straw, flax straw, kenaf, industrial hemp, sisal, textile flat straw, hesperaloe, etc. Thus, the present invention also encompasses the use of the perhydrolases of the present invention in pulp treatment methods.

As used herein, “oxidizing chemical” refers to a chemical that has the capability of bleaching pulp or any other material. The oxidizing chemical is present at an amount, pH and temperature suitable for bleaching. The term includes, but is not limited to hydrogen peroxide and peracids.

As used herein, “acyl” is the general name for organic acid groups, which are the residues of carboxylic acids after removal of the —OH group (e.g., ethanoyl chloride, CH₃CO—Cl, is the acyl chloride formed from ethanoic acid, CH₃COO—H). The names of the individual acyl groups are formed by replacing the “-ic” of the acid by “-yl.”

As used herein, the term “acylation” refers to the chemical transformation which substitutes the acyl (RCO—) group into a molecule, generally for an active hydrogen of an —OH group.

As used herein, the term “transferase” refers to an enzyme that catalyzes the transfer of functional compounds to a range of substrates.

As used herein, “leaving group” refers to the nucleophile which is cleaved from the acyl donor upon substitution by another nucleophile.

As used herein, the term “enzymatic conversion” refers to the modification of a substrate to an intermediate or the modification of an intermediate to an end-product by contacting the substrate or intermediate with an enzyme. In some embodiments, contact is made by directly exposing the substrate or intermediate to the appropriate enzyme. In other embodiments, contacting comprises exposing the substrate or intermediate to an organism that expresses and/or excretes the enzyme, and/or metabolizes the desired substrate and/or intermediate to the desired intermediate and/or end-product, respectively.

As used herein, the phrase “detergent stability” refers to the stability of a detergent composition. In some embodiments, the stability is assessed during the use of the detergent, while in other embodiments, the term refers to the stability of a detergent composition during storage.

As used herein, the phrase, “stability to proteolysis” refers to the ability of a protein (e.g., an enzyme) to withstand proteolysis. It is not intended that the term be limited to the use of any particular protease to assess the stability of a protein.

As used herein, “oxidative stability” refers to the ability of a protein to function under oxidative conditions. In particular, the term refers to the ability of a protein to function in the presence of various concentrations of H₂O₂ and/or peracid. Stability under various oxidative conditions can be measured either by standard procedures known to those in the art and/or by the methods described herein. A substantial change in oxidative stability is evidenced by at least about a 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity present in the absence of oxidative compounds.

As used herein, “pH stability” refers to the ability of a protein to function at a particular pH. In general, most enzymes have a finite pH range at which they will function. In addition to enzymes that function in mid-range pHs (i.e., around pH 7), there are enzymes that are capable of working under conditions with very high or very low pHs. Stability at various pHs can be measured either by standard procedures known to those in the art and/or by the methods described herein. A substantial change in pH stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity at the enzyme's optimum pH. However, it is not intended that the present invention be limited to any pH stability level nor pH range.

As used herein, “thermal stability” refers to the ability of a protein to function at a particular temperature. In general, most enzymes have a finite range of temperatures at which they will function. In addition to enzymes that work in mid-range temperatures (e.g., room temperature), there are enzymes that are capable of working in very high or very low temperatures. Thermal stability can be measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the catalytic activity of a mutant when exposed to a different temperature (i.e., higher or lower) than optimum temperature for enzymatic activity. However, it is not intended that the present invention be limited to any temperature stability level nor temperature range.

As used herein, the term “chemical stability” refers to the stability of a protein (e.g., an enzyme) towards chemicals that adversely affect its activity. In some embodiments, such chemicals include, but are not limited to hydrogen peroxide, peracids, anionic detergents, cationic detergents, non-ionic detergents, chelants, etc. However, it is not intended that the present invention be limited to any particular chemical stability level nor range of chemical stability.

As used herein, the phrase “perhydrolase activity improvement” refers to the relative improvement of perhydrolase activity, in comparison with a standard enzyme. In some embodiments, the term refers to an improved rate of perhydrolysis product, while in other embodiments, the term encompasses perhydrolase compositions that produce less hydrolysis product. In additional embodiments, the term refers to perhydrolase compositions with altered substrate specificity.

As used herein, the phrase “alteration in substrate specificity” refers to changes in the substrate specificity of an enzyme. In some embodiments, a change in substrate specificity is defined as a difference between the K_(cat)/K_(m) ratio observed with an enzyme compared to enzyme variants or other enzyme compositions. Enzyme substrate specificities vary, depending upon the substrate tested. The substrate specificity of an enzyme is determined by comparing the catalytic efficiencies it exhibits with different substrates. These determinations find particular use in assessing the efficiency of mutant enzymes, as it is generally desired to produce variant enzymes that exhibit greater ratios for particular substrates of interest. For example, the perhydrolase enzymes of the present invention are more efficient in producing peracid from an ester substrate than enzymes currently being used in cleaning, bleaching and disinfecting applications. Another example of the present invention is a perhydrolase with a lower activity on peracid degradation compared to the wild type. Another example of the present invention is a perhydrolase with higher activity on more hydrophobic acyl groups than acetic acid. However, it is not intended that the present invention be limited to any particular substrate composition nor any specific substrate specificity.

As used herein, “surface property” is used in reference to an electrostatic charge, as well as properties such as the hydrophobicity and/or hydrophilicity exhibited by the surface of a protein.

As used herein, the phrase “is independently selected from the group consisting of . . . ” means that moieties or elements that are selected from the referenced Markush group can be the same, can be different or any mixture of elements as indicated in the following example:

A molecule having 3 R groups wherein each R group is independently selected from the group consisting of A, B and C. Here the three R groups may be: AAA, BBB, CCC, AAB, AAC, BBA, BBC, CCA, CCB, or ABC.

In reference to chemical compositions, the term “substituted” as used herein, means that the organic composition or radical to which the term is applied is:

-   -   (a) made unsaturated by the elimination of at least one element         or radical; or     -   (b) at least one hydrogen in the compound or radical is replaced         with a moiety containing one or more (i) carbon, (ii)         oxygen, (iii) sulfur, (iv) nitrogen or (v) halogen atoms; or     -   (c) both (a) and (b).         Moieties which may replace hydrogen as described in (b)         immediately above, that contain only carbon and hydrogen atoms,         are hydrocarbon moieties including, but not limited to, alkyl,         alkenyl, alkynyl, alkyldienyl, cycloalkyl, phenyl, alkyl phenyl,         naphthyl, anthryl, phenanthryl, fluoryl, steroid groups, and         combinations of these groups with each other and with polyvalent         hydrocarbon groups such as alkylene, alkylidene and alkylidyne         groups. Moieties containing oxygen atoms that may replace         hydrogen as described in (b) immediately above include, but are         not limited to, hydroxy, acyl or keto, ether, epoxy, carboxy,         and ester containing groups. Moieties containing sulfur atoms         that may replace hydrogen as described in (b) immediately above         include, but are not limited to, the sulfur-containing acids and         acid ester groups, thioether groups, mercapto groups and         thioketo groups. Moieties containing nitrogen atoms that may         replace hydrogen as described in (b) immediately above include,         but are not limited to, amino groups, the nitro group, azo         groups, ammonium groups, amide groups, azido groups, isocyanate         groups, cyano groups and nitrile groups. Moieties containing         halogen atoms that may replace hydrogen as described in (b)         immediately above include chloro, bromo, fluoro, iodo groups and         any of the moieties previously described where a hydrogen or a         pendant alkyl group is substituted by a halo group to form a         stable substituted moiety.

It is understood that any of the above moieties (b)(i) through (b)(v) can be substituted into each other in either a monovalent substitution or by loss of hydrogen in a polyvalent substitution to form another monovalent moiety that can replace hydrogen in the organic compound or radical.

As used herein, the terms “purified” and “isolated” refer to the removal of contaminants from a sample. For example, perhydrolases are purified by removal of contaminating proteins and other compounds within a solution or preparation that are not perhydrolases. In some embodiments, recombinant perhydrolases are expressed in bacterial or fungal host cells and these recombinant perhydrolases are purified by the removal of other host cell constituents; the percent of recombinant perhydrolase polypeptides is thereby increased in the sample.

As used herein, “protein of interest,” refers to a protein (e.g., an enzyme or “enzyme of interest”) which is being analyzed, identified and/or modified. Naturally-occurring, as well as recombinant proteins find use in the present invention.

As used herein, “protein” refers to any composition comprised of amino acids and recognized as a protein by those of skill in the art. The terms “protein,” “peptide” and polypeptide are used interchangeably herein. Wherein a peptide is a portion of a protein, those skilled in the art understand the use of the term in context.

As used herein, functionally and/or structurally similar proteins are considered to be “related proteins.” In some embodiments, these proteins are derived from a different genus and/or species, including differences between classes of organisms (e.g., a bacterial protein and a fungal protein). In some embodiments, these proteins are derived from a different genus and/or species, including differences between classes of organisms (e.g., a bacterial enzyme and a fungal enzyme). In additional embodiments, related proteins are provided from the same species. Indeed, it is not intended that the present invention be limited to related proteins from any particular source(s). In addition, the term “related proteins” encompasses tertiary structural homologs and primary sequence homologs (e.g., the perhydrolase of the present invention). In further embodiments, the term encompasses proteins that are immunologically cross-reactive. In most particularly preferred embodiments, the related proteins of the present invention very high ratios of perhydrolysis to hydrolysis.

As used herein, the term “derivative” refers to a protein which is derived from a protein by addition of one or more amino acids to either or both the C- and N-terminal end(s), substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, and/or deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, and/or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of a protein derivative is preferably achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative protein.

Related (and derivative) proteins comprise “variant proteins.” In some preferred embodiments, variant proteins differ from a parent protein and one another by a small number of amino acid residues. The number of differing amino acid residues may be one or more, preferably 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In some preferred embodiments, the number of different amino acids between variants is between 1 and 10. In some particularly preferred embodiments, related proteins and particularly variant proteins comprise at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, a related protein or a variant protein as used herein, refers to a protein that differs from another related protein or a parent protein in the number of prominent regions. For example, in some embodiments, variant proteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions that differ from the parent protein.

Several methods are known in the art that are suitable for generating variants of the perhydrolase enzymes of the present invention, including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.

In particularly preferred embodiments, homologous proteins are engineered to produce enzymes with the desired activity(ies). In some particularly preferred embodiments, the engineered proteins are included within the SGNH-hydrolase family of proteins. In some most preferred embodiments, the engineered proteins comprise at least one or a combination of the following conserved residues: L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205. In alternative embodiments, these engineered proteins comprise the GDSL-GRTT and/or ARTT motifs. In further embodiments, the enzymes are multimers, including but not limited to dimers, octamers, and tetramers. In yet additional preferred embodiments, the engineered proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than 1.

An amino acid residue of a perhydrolase is equivalent to a residue of M. smegmatis perhydrolase if it is either homologous (i.e., having a corresponding position in either the primary and/or tertiary structure) or analogous to a specific residue or portion of that residue in M. smegmatis perhydrolase (i.e., having the same or similar functional capacity to combine, react, and/or chemically interact).

In some embodiments, in order to establish homology to primary structure, the amino acid sequence of a perhydrolase is directly compared to the M. smegmatis perhydrolase primary sequence and particularly to a set of residues known to be invariant in all perhydrolases for which sequence is known. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of M. smegmatis perhydrolase are defined. In preferred embodiments, alignment of conserved residues conserves 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues are also adequate to define equivalent residues. In preferred embodiments, conservation of the catalytic serine and histidine residues are maintained. Conserved residues are used to define the corresponding equivalent amino acid residues of M. smegmatis perhydrolase in other perhydrolases (e.g., perhydrolases from other Mycobacterium species, as well as any other organisms).

In some embodiments of the present invention, the DNA sequence encoding M. smegmatis perhydrolase is modified. In some embodiments, the following residues are modified: Cys7, Asp10, Ser1, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194, and Phe196. However, it is not intended that the present invention be limited to sequence that are modified at these positions. Indeed, it is intended that the present invention encompass various modifications and combinations of modifications.

In additional embodiments, equivalent residues are defined by determining homology at the level of tertiary structure for a perhydrolase whose tertiary structure has been determined by x-ray crystallography. In this context, “equivalent residues” are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the carbonyl hydrolase and M. smegmatis perhydrolase (N on N, CA on CA, C on C, and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the perhydrolase in question to the M. smegmatis perhydrolase. As known in the art, the best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. Equivalent residues which are functionally and/or structurally analogous to a specific residue of M. smegmatis perhydrolase are defined as those amino acids of the perhydrolases that preferentially adopt a conformation such that they either alter, modify or modulate the protein structure, to effect changes in substrate binding and/or catalysis in a manner defined and attributed to a specific residue of the M. smegmatis perhydrolase. Further, they are those residues of the perhydrolase (in cases where a tertiary structure has been obtained by x-ray crystallography), which occupy an analogous position to the extent that although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of M. smegmatis perhydrolase. The coordinates of the three dimensional structure of M. smegmatis perhydrolase were determined and are set forth herein (See e.g., Example 14) and find use as outlined above to determine equivalent residues on the level of tertiary structure.

In some embodiments, some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. The perhydrolase mutants of the present invention include various mutants, including those encoded by nucleic acid that comprises a signal sequence. In some embodiments of perhydrolase mutants that are encoded by such a sequence are secreted by an expression host. In some further embodiments, the nucleic acid sequence comprises a homolog having a secretion signal.

Characterization of wild-type and mutant proteins is accomplished via any means suitable and is preferably based on the assessment of properties of interest. For example, pH and/or temperature, as well as detergent and or oxidative stability isare determined in some embodiments of the present invention. Indeed, it is contemplated that enzymes having various degrees of stability in one or more of these characteristics (pH, temperature, proteolytic stability, detergent stability, and/or oxidative stability) will find use. In still other embodiments, perhydrolases with low peracid degradation activity are selected.

As used herein, “expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid,” “expression plasmid,” and “vector” are often used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.

In some preferred embodiments, the perhydrolase gene is ligated into an appropriate expression plasmid. The cloned perhydrolase gene is then used to transform or transfect a host cell in order to express the perhydrolase gene. This plasmid may replicate in hosts in the sense that it contains the well-known elements necessary for plasmid replication or the plasmid may be designed to integrate into the host chromosome. The necessary elements are provided for efficient gene expression (e.g., a promoter operably linked to the gene of interest). In some embodiments, these necessary elements are supplied as the gene's own homologous promoter if it is recognized, (i.e., transcribed, by the host), a transcription terminator (a polyadenylation region for eukaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the perhydrolase gene. In some embodiments, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing media is also included.

The following cassette mutagenesis method may be used to facilitate the construction of the perhydrolase variants of the present invention, although other methods may be used.

First, as described herein, a naturally-occurring gene encoding the perhydrolase is obtained and sequenced in whole or in part. Then, the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded perhydrolase. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protein gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the perhydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.

Once the naturally-occurring DNA and/or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

As used herein, “corresponding to,” refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.

As used herein, “corresponding region,” generally refers to an analogous position along related proteins or a parent protein.

The terms “nucleic acid molecule encoding,” “nucleic acid sequence encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

As used herein, the term “analogous sequence” refers to a sequence within a protein that provides similar function, tertiary structure, and/or conserved residues as the protein of interest (i.e., typically the original protein of interest). For example, in epitope regions that contain an alpha helix or a beta sheet structure, the replacement amino acids in the analogous sequence preferably maintain the same specific structure. The term also refers to nucleotide sequences, as well as amino acid sequences. In some embodiments, analogous sequences are developed such that the replacement amino acids result in a variant enzyme showing a similar or improved function. In some preferred embodiments, the tertiary structure and/or conserved residues of the amino acids in the protein of interest are located at or near the segment or fragment of interest. Thus, where the segment or fragment of interest contains, for example, an alpha-helix or a beta-sheet structure, the replacement amino acids preferably maintain that specific structure.

As used herein, “homologous protein” refers to a protein (e.g., perhydrolase) that has similar action and/or structure, as a protein of interest (e.g., an perhydrolase from another source). It is not intended that homologs be necessarily related evolutionarily. Thus, it is intended that the term encompass the same or similar enzyme(s) (i.e., in terms of structure and function) obtained from different species. In some preferred embodiments, it is desirable to identify a homolog that has a quaternary, tertiary and/or primary structure similar to the protein of interest, as replacement for the segment or fragment in the protein of interest with an analogous segment from the homolog will reduce the disruptiveness of the change. In some embodiments, homologous proteins have induce similar immunological response(s) as a protein of interest.

As used herein, “homologous genes” refers to at least a pair of genes from different species, which genes correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes). These genes encode “homologous proteins.”

As used herein, “ortholog” and “orthologous genes” refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.

As used herein, “paralog” and “paralogous genes” refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species.

As used herein, “wild-type” and “native” proteins are those found in nature. The terms “wild-type sequence,” and “wild-type gene” are used interchangeably herein, to refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project. The genes encoding the naturally-occurring protein may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protein of interest, preparing genomic libraries from organisms expressing the protein, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The term “recombinant DNA molecule” as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.

The term “recombinant oligonucleotide” refers to an oligonucleotide created using molecular biological manipulations, including but not limited to, the ligation of two or more oligonucleotide sequences generated by restriction enzyme digestion of a polynucleotide sequence, the synthesis of oligonucleotides (e.g., the synthesis of primers or oligonucleotides) and the like.

The degree of homology between sequences may be determined using any suitable method known in the art (See e.g., Smith and Waterman, Adv. Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT, FAS TA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.); and Devereux et al., Nucl. Acid Res., 12:387-395 [1984]).

For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol., 35:351-360 [1987]). The method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al., (Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). One particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol., 266:460-480 [1996]). parameters “W,” “T,” and “X” determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]) alignments (B) of 50, expectation (E) of 10, M′S, N′-4, and a comparison of both strands.

As used herein, “percent (%) nucleic acid sequence identity” is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of the sequence.

As used herein, the term “hybridization” refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.

As used herein, the phrase “hybridization conditions” refers to the conditions under which hybridization reactions are conducted. These conditions are typically classified by degree of “stringency” of the conditions under which hybridization is measured. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm-5° C. (5° below the Tm of the probe); “high stringency” at about 5-10° below the Tm; “intermediate stringency” at about 10-20° below the Tm of the probe; and “low stringency” at about 20-25° below the Tm. Alternatively, or in addition, hybridization conditions can be based upon the salt or ionic strength conditions of hybridization and/or one or more stringency washes. For example, 6×SSC=very low stringency; 3×SSC=low to medium stringency; 1×SSC=medium stringency; and 0.5×SSC=high stringency. Functionally, maximum stringency conditions may be used to identify nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe.

For applications requiring high selectivity, it is typically desirable to use relatively stringent conditions to form the hybrids (e.g., relatively low salt and/or high temperature conditions are used).

The phrases “substantially similar and “substantially identical” in the context of at least two nucleic acids or polypeptides typically means that a polynucleotide or polypeptide comprises a sequence that has at least about 40% identity, more preferable at least about 50% identity, yet more preferably at least about 60% identity, preferably at least about 75% identity, more preferably at least about 80% identity, yet more preferably at least about 90%, still more preferably about 95%, most preferably about 97% identity, sometimes as much as about 98% and about 99% sequence identity, compared to the reference (i.e., wild-type) sequence. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See e.g., Altschul, et al., J. Mol. Biol. 215:403-410 [1990]; Henikoff et al., Proc. Natl. Acad. Sci. USA 89:10915 [1989]; Karin et al., Proc. Natl. Acad. Sci. USA 90:5873 [1993]; and Higgins et al., Gene 73:237-244 [1988]). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448 [1988]). One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

As used herein, “equivalent residues” refers to proteins that share particular amino acid residues. For example, equivalent resides may be identified by determining homology at the level of tertiary structure for a protein (e.g., perhydrolase) whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the protein having putative equivalent residues and the protein of interest (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins analyzed. The preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art of crystallography and protein characterizationanalysis.

As used herein, the terms “hybrid perhydrolases” and “fusion perhydrolases” refer to proteins that are engineered from at least two different or “parental” proteins. In preferred embodiments, these parental proteins are homologs of one another. For example, in some embodiments, a preferred hybrid perhydrolase or fusion protein contains the N-terminus of a protein and the C-terminus of a homolog of the protein. In some preferred embodiment, the two terminal ends are combined to correspond to the full-length active protein.

The term “regulatory element” as used herein refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals and termination signals.

As used herein, “host cells” are generally prokaryotic or eukaryotic hosts which are transformed or transfected with vectors constructed using recombinant DNA techniques known in the art. Transformed host cells are capable of either replicating vectors encoding the protein variants or expressing the desired protein variant. In the case of vectors which encode the pre- or prepro-form of the protein variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.

The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means transformation, transduction or transfection. Means of transformation include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA and the like as known in the art. (See, Chang and Cohen, Mol. Gen. Genet., 168:111-115 [1979]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; and the review article by Ferrari et al., in Harwood, Bacillus, Plenum Publishing Corporation, pp. 57-72 [1989]).

The term “promoter/enhancer” denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (for example, the long terminal repeats of retroviruses contain both promoter and enhancer functions). The enhancer/promoter may be “endogenous” or “exogenous” or “heterologous.” An endogenous enhancer/promoter is one which is naturally linked with a given gene in the genome. An exogenous (heterologous) enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques).

The presence of “splicing signals” on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.7-16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

The term “stable transfection” or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term “stable transfectant” refers to a cell which has stably integrated foreign or exogenous DNA into the genomic DNA of the transfected cell.

The terms “selectable marker” or “selectable gene product” as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.

As used herein, the terms “amplification” and “gene amplification” refer to a process by which specific DNA sequences are disproportionately replicated such that the amplified gene becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both. Selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) may result in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

As used herein, the term “co-amplification” refers to the introduction into a single cell of an amplifiable marker in conjunction with other gene sequences (i.e., comprising one or more non-selectable genes such as those contained within an expression vector) and the application of appropriate selective pressure such that the cell amplifies both the amplifiable marker and the other, non-selectable gene sequences. The amplifiable marker may be physically linked to the other gene sequences or alternatively two separate pieces of DNA, one containing the amplifiable marker and the other containing the non-selectable marker, may be introduced into the same cell.

As used herein, the terms “amplifiable marker,” “amplifiable gene,” and “amplification vector” refer to a marker, gene or a vector encoding a gene which permits the amplification of that gene under appropriate growth conditions.

As used herein, the term “amplifiable nucleic acid” refers to nucleic acids which may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”

As used herein, the term “sample template” refers to nucleic acid originating from a sample which is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

“Template specificity” is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q13 replicase, MDV-1 RNA is the specific template for the replicase (See e.g., Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (See, Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (See, Wu and Wallace, Genomics 4:560 [1989]). Finally, Tag and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences.

As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the term “target,” when used in reference to amplification methods (e.g., the polymerase chain reaction), refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the “target” is sought to be sorted out from other nucleic acid sequences. A “segment” is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

The Present Invention

In some most particularly preferred embodiments, the present invention finds use in the enzymatic generation of peracids from ester substrates and hydrogen peroxide. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Importantly, the present invention provides means for effective cleaning, bleaching, and disinfecting over broad pH and temperature ranges. In some embodiments, the pH range utilized in this generation is 4-12. In alternative embodiments, the temperature range utilized is between 5° and 90° C. The present invention provides advantages over the presently used systems (See e.g., EP Appln. 87-304933.9) in that bleaching is possible at the optimum pH of peracid oxidation, as well as providing bleaching at neutral pH, acidic pHs, and at low temperatures. While the present invention is described herein most fully in regard to laundry and fabric care, it is not intended that the present invention be limited to these applications. Indeed, the present invention finds use in various settings, particularly those in which bleaching by peracids and/or hydrogen peroxide are desired, including but not limited to laundry, fabric treatment, pulp and paper processing, personal care applications, disinfection and cleaning of hard surfaces. For example, it is contemplated that the compositions of the present invention will find use in bleaching of pulp, including use in methods such as those set forth in U.S. Pat. Nos. 6,569,286, 5,785,812, 6,165,318, and 4,400,237, all of which are herein incorporated by reference.

Historically, sodium perborate, and more recently, sodium percarbonate, have been used as bleaching compounds, particularly in European laundry detergents. This compound decomposes rapidly in aqueous solution to yield hydrogen peroxide (H₂O₂), which is the active bleaching species. As sodium perborate is more active at temperatures above 80° C., and less active in the temperature range of 40-60° C. (i.e., wash temperatures that have become most commonly preferred as of the 1950s), bleaching activators have been incorporated into laundry detergents that contain sodium perborate. Indeed, most laundry detergents contain bleaching activators. These activators are compounds with O- or N-bounded acetyl groups that are able to react with the strongly nucleophilic hydroperoxy anion to yield peroxyacetic acid. Since the reacting species is hydroperoxy anion, alkaline pHs are essential for the efficient conversion of these activators to peracids. The peroxyacetic acid is decomposed in weakly basic media to form singlet oxygen (See, Hofmann et al., J. Prakt. Chem., 334:293-297 [1992]).

Hydrogen peroxide is a particularly effective bleach at high temperatures (e.g., >40° C.) and pH (>10), conditions that are typically used in washing fabrics in some settings. However, as indicated above, cold water washing is becoming more commonly used and results in less effective bleaching by H₂O₂ than use of hot water. To overcome this low temperature disadvantage, detergent formulations typically include bleach boosters, such as TAED (N,N,N′N′-tetraacetylethylenediamine), NOBS (nonanoyloxybenzene sulfonate), etc. These boosters combine with H₂O₂ to form peracetic acid, a peracid species that is more effective than H₂O₂ alone. Although it helps the bleaching capability of detergent, the TAED reaction is only approximately 50% efficient, as only two out of the four acetyl groups in TAED are converted to peracids. Additionally, conversion of TAED into peracetic acid by hydrogen peroxide is efficient only at alkaline pHs and high temperatures. Thus, the TAED reaction is not optimized for use in all bleaching applications (e.g., those involving neutral or acidic pHs, and cold water). The present invention provides means to overcome the disadvantages of TAED use. For example, the present invention finds use in cold water applications, as well as those involving neutral or acidic pH levels. Furthermore, the present invention provides means for peracid generation from hydrogen peroxide, with a high perhydrolysis to hydrolysis ratio. The present invention further provides advantages over compositions that contain enzymes such as esterases and lipases) which have very low perhydrolysis to hydrolysis ratios.

In addition to its applications in detergents, the present invention provides methods and compositions for the use of peracids in textile bleaching and in various other applications. In some embodiments, the present invention provides one-step methods for textile processing applications, including but not limited to one-step desizing, scouring and bleaching processes (See e.g., EP WO 03002810, EP 1255888, WO 0164993, and US 20020007516, all of which are hereby incorporated by reference). As described in greater detail herein, in some embodiments, bleaching involves processing textile material before it is dyed and/or after it is incorporated into textile goods. However, it is not intended that the present invention be limited to any particular regimen of use nor any particular textile material.

Furthermore, the peracetic technology of the present invention finds use as an effective bactericide (See, Baldry, J. Appl. Bacteriol., 54:417-423 [1983]). Thus, the present invention provides compositions and methods for the sterilization/disinfection of various objects, including but not limited to medical devices, medical equipment, industrial equipment, and fermenters, as well as any additional object that needs to be sterilized or disinfected. As discussed in greater detail below, during the development of the present invention, the enzyme of the present invention was used in a standard cell kill experiment to demonstrate this suitability. In additional embodiments, the present invention provides compositions and methods suitable for use in biofilm control, such as in cooling towers.

Also as described in more detail in the Examples below, the present invention provides many advantages for cleaning and/or sterilization of a wide range of objects, including but not limited to clothing, fabrics, medical devices, etc. In addition, the present invention provides compositions that are effective in cleaning, bleaching, and disinfecting, over a range of wash temperatures and pHs. In additional embodiments, the present invention finds use in degradation of peracids through the perhydrolase peracid degradation activity. In some preferred embodiments, this activity is used in peracid waste clean up applications.

Furthermore, the perhydrolase enzymes of the present invention are active on various acyl donor substrates, as well as being active at low substrate concentrations, and provide means for efficient perhydrolysis due to the high peracid:acid ratio. Indeed, it has been recognized that higher perhydrolysis to hydrolysis ratios are preferred for bleaching applications (See e.g., U.S. Pat. Nos. 5,352,594, 5,108,457, 5,030,240, 3974,082, and 5,296,616, all of which are herein incorporated by reference). In preferred embodiments, the perhydrolase enzymes of the present invention provide perhydrolysis to hydrolysis ratios that are greater than 1. In particularly preferred embodiments, the perhydrolase enzymes provide a perhydrolysis to hydrolysis ratio greater than 1 and are find use in bleaching.

In addition, it has been shown to be active in commonly used detergent formulations (e.g., Aria Futur, WOB, etc.). Thus, the present invention provides many advantages in various cleaning settings.

As indicated above, key components to peracid production by enzymatic perhydrolysis are enzyme, ester substrate, and hydrogen peroxide. Hydrogen peroxide can be either added directly in batch, or generated continuously “in situ.” Current washing powders use batch additions of H₂O₂, in the form of percarbonate or perborate salts that spontaneously decompose to H₂O₂. The perhydrolase enzymes of the present invention find use in the same washing powder batch method as the H₂O₂ source. However, these enzymes also find use with any other suitable source of H₂O₂, including that generated by chemical, electro-chemical, and/or enzymatic means. Examples of chemical sources are the percarbonates and perborates mentioned above, while an example of an electrochemical source is a fuel cell fed oxygen and hydrogen gas, and an enzymatic example includes production of H₂O₂ from the reaction of glucose with glucose oxidase. The following equation provides an example of a coupled system that finds use with the present invention.

It is not intended that the present invention be limited to any specific enzyme, as any enzyme that generates H₂O₂ with a suitable substrate finds use in the methods of the present invention. For example, lactate oxidases from Lactobacillus species which are known to create H₂O₂ from lactic acid and oxygen find use with the present invention. Indeed, one advantage of the methods of the present invention is that the generation of acid (e.g., gluconic acid in the above example) reduces the pH of a basic solution to the pH range in which the peracid is most effective in bleaching (i.e., at or below the pKa). Other enzymes (e.g., alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase, etc.) that can generate hydrogen peroxide also find use with ester substrates in combination with the perhydrolase enzymes of the present invention to generate peracids. In some preferred embodiments, the ester substrates are selected from one or more of the following acids: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, as described herein, the present invention provides definite advantages over the currently used methods and compositions for detergent formulation and use, as well as various other applications.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

Cloning and Characterization of M. smegmatis Perhydrolase

The cloning of the M. smegmatis perhydrolase (i.e., referred to herein as the “phd” gene, which encodes the “Phd” protein; this perhydrolase gene is sometimes herein referred to as the “act” gene and the protein is sometimes referred to as the “Act” protein) of the present invention was based on peptide sequence data from the acyltransferase purified from Mycobacterium parafortuitum (previously known as Corynebacterium oxydans) and published information regarding the 7-aminocephalosporanic acid (7-ACA) arylesterase gene of Agrobacterium radiobacter (Sakai et al., J. Ferment. Bioengineer., 85: 138-143 [1998]). Two peptide sequences from purified M. parafortuitum acyltransferase were found to be similar to internal N- and C-terminal regions of the A. radiobacter 7-ACA-arylesterase (47% and 42% identity respectively).

A set of PCR primers was designed based on the amino acid sequence of these internal peptides (designated “AtintF” and “AtintR”). Another set of primers was developed based on the 5′ and 3′ ends (“ATNcoI” and “ATBamH1”) of theA. radiobacter 7-ACA DNA sequence. A single product of the expected size was amplified from M. parafortuitum chromosomal DNA using both sets of primers. The full length product, amplified by the ATNcoI/ATBamH1 primer pair, was cloned into pET16b and transformed into BL21 cells (Novagen, Madison, Wis.). This clone had a sequence identical to that of the A. radiobacter 7-ACA gene. As it was determined that purified M. parafortuitum perhydrolase was not the 7-ACA acyl esterase, it was concluded that this was not the gene encoding the perhydrolase of the present invention.

Thus, efforts were further focused on M. smegmatis for cloning and expression of the perhydrolase of the present invention. To identify the M. parafortuitum gene based on enzyme activity screening, a plasmid library of M. parafortuitum DNA in M. smegmatis was constructed using a plasmid with a promoter to drive expression of cloned genes. Surprisingly, M. smegmatis itself was found to be positive for perhydrolase and acyltransferase activity. Thus, in some instances herein, the perhydrolase is referred to as “ACT” (or “Act”). A protein BLAST search of the M. smegmatis unfinished genome using the sequence of the A. radiobacter 7-ACA identified a 2 kb contig containing an ORF (open reading frame) that encoded a hypothetical protein that was similar but not identical to the 7-ACA protein. Based on this sequence, primers were designed and used to amplify the gene from M. smegmatis (ATCC 10143). By adding an E. coli ribosome binding site upstream of the start codon, a clone that expressed active enzyme was obtained. The vector used was either pCR2.1TOPO or pBluntIITOPO (Invitrogen, Carlsbad, Calif.), in E. coli Top10 cells. The gene was expressed constitutively from the plasmid-encoded lac promoter. This enzyme carried out the same reactions as the originally described M. parafortuitum acyltransferase.

During the characterization of the perhydrolase of the present invention, standard protein BLAST searches identified a few proteins (<20) with sequence similarity of 30-80%. This group included the 7-ACA arylesterases from A. radiobacter and other organisms, which have 43% identity with M. smegmatis perhydrolase. All of the identified homologs with at least 40% similarity have a GDS motif very near the N-terminal end. All of the proteins also contain most of the conserved residues which could place them within the suggested GDSL family of lipolytic enzymes (See e.g., Upton and Buckley, Trends Biochem. Sci., 20:178 [1995]). However, enzymes mentioned in this paper do not appear on homology searches with the perhydrolase protein. Indeed these proteins have less than 20% similarity with the perhydrolase and its homologs, suggesting that the acyltransferase-related (and perhydrolase of the present invention) enzymes form a subfamily.

The natural function of the enzyme of the present invention and the closely related proteins, apart from the 7-ACA arylesterase, have not been biochemically determined M. smegmatis appears to be the only organism with the acyltransferaseperhydrolase in an operon with a putative penicillin binding protein (PBP). While it is not intended that the present invention be limited to any particular mechanism, this suggests that the enzyme may be involved in cell wall synthesisstructure or modification of molecules taken up from the environment. There are no homologues of the perhydrolase of the present invention that have been identified in M. tuberculosis or M. leprae to date. However, some organisms were determined to have multiple homologues (e.g., S. meliloti).

During the development of the present invention, various mutations were made in the M. smegmatis perhydrolase in order to assess its activity. This enzyme contains two cysteine residues, which were hypothesized as potentially forming disulfide bonds, both of which were changed to alanine, in order to determine whether or not the C residues had any effect on the activity of the enzyme. Activity assay results obtained using the transesterification (in aqueous solution) assay described herein indicated that C7A, as well as C77A, and a double mutant (C7A and C77A) were of the same size and specific activity.

Many enzymes have the amino acid serine as part of their active site and are therefore referred to, among other designations, as “serine hydrolases.” The active site may consist of a catalytic triad of S (serine), D (aspartic acid) and H (histidine). Examples of such enzymes include, but are not limited to subtilisin (D32-H64-S215), chymotrypsin (H57-D102-5195) and lipases in the alphabeta hydrolase family (e.g., S126-D176-H206). A typical motif for lipases is the GDSL motif (Upton and Buckley, supra [1995]) in which the S is the active site serine. Since the perhydrolase of the present invention was determined to have a GDSL (amino acids 9-12) motif, the S11 was mutated to an A, in order to confirm the involvement of this S in the active site. As indicated in the Examples, the activity assay results indicated that S11A had only 1% of the activity of the wild-type enzyme. Deletion of the C-terminal 25 amino acids also resulted in abrogation of the activity, suggesting that these amino acids either contained a residue involved directly in the active site, and/or that the structure of the protein was affected such that the active site was no longer able to catalyze the reactions. In addition, the predicted active site residues, D192 and H195 were mutated to A. Neither mutant had activity, confirming that the active site residues of the perhydrolase of the present invention consist of S11, D192 and H195. However, it is not intended that the present invention be limited to any particular mechanism, nor is the present invention limited to mutation(s) at any particular active site residues.

Cloning of M. parafortuitum Perhydrolase

There were some differences between the N-terminal peptide sequence obtained from the M. parafortuitum enzyme and the N-terminal sequence of M. smegmatis perhydrolase. However, there was a sequence in the C-terminal region of the M. smegmatis perhydrolase identical to the C-terminal peptide sequence of the M. parafortuitum enzyme. Two primers were designed to amplify a partial sequence of the M. parafortuitum perhydrolase gene; the sequence of the reverse primer was identical to the sequence of the corresponding region in M. smegmatis perhydrolase gene, and the sequence of the forward primer was based on M. smegmatis codon usage. The forward primer, MPS: 5′-ATGGGTACCCGACGAATTCTGTCCTTCGGTGATTCCCTGACCT-3′ (SEQ ID NO:11) and the reverse primer MPC-intR 5′-GATTCCGTCGACGCCGTCGGTGCTGATCACCGAACCCGCGTCGAAGAACGG-3′ (SEQ ID NO:12). The partial gene was amplified from the chromosome of M. parafortuitum and cloned into pCR2.1TOPO (Invitrogen, Carlsbad, Calif.). Sequence analysis showed that the enzyme is very similar, but not identical to the M. smegmatis perhydrolase (77% identity). Based on the molecular weights of the monomers of the perhydrolases determined by SDS-PAGE (MP AT: 26 kDa, MSAT: 24 kDa, MP cloned AT: ˜18 kDa), the clone from primers made to the internal fragment was determined to be missing approximately 70 amino acids (˜8 kDa). The remaining sequence at the 5′-end of the M. parafortuitum gene can be obtained by any of several methods suitable and familiar to those skilled in the art of molecular biology, including, but not limited to, inverse PCR, probing of plasmidcosmid libraries of M. parafortuitum chromosomal DNA, sequencing of the gene directly from chromosomal DNA (e.g., as performed by Fidelity Systems, Bethesda Md.).

Expression of the M. smegmatis Perhydrolase

The perhydrolase is an intracellular protein in its native host. Production of the perhydrolase in non-native hosts may also be done intracellularly. However, in some embodiments, a signal sequence is added to the perhydrolase, which facilitates expression of the perhydrolase by secretion into the periplasm (i.e., in Gram-negative organisms, such as E. coli), or into the extracellular space (i.e., in Gram-positive organisms, such as Bacillus and Actinomycetes), or eukaryotic hosts (e.g., Trichoderma, Aspergillus, Saccharomyces, and Pichia). Of course, these are just a few examples of possible prokaryotic and eukaryotic hosts. It is not intended that the present invention be limited to these specific hosts, as various other organisms find use as expression hosts in the present invention.

A variety of commercially available expression systems, including but not limited to pBAD, plac, T7, find use in the expression of the perhydrolase in Gram-negative hosts (e.g., E. coli). In some embodiments, the same types of promoters find use in another Gram-negative host, Pantoea citrea.

To test expression in E. coli two strategies were used: 1) adding an RBS (ribosome binding site) to the 5′ end of the phd gene and cloning the gene into pCRBLUNTIITOPO (Invitrogen), thus allowing expression directly from the pLac promoter available in that vector; and 2) cloning the phd gene under control of the T7 promoter in the plasmid pET16b (Novagen). In the latter system, expression of the gene is inducible by addition of IPTG to the growing culture and use of a specific host cell (e.g., BL21(λDE3)pLysS (Novagen)) that contains the 2DE3 lysogen encoding the T7 RNA polymerase. The first strategy produces a plasmid capable of allowing expression of the perhydrolase protein in other Gram-negative hosts (e.g., P. citrea).

To express protein in E. coli or P. citrea using the first strategy, cultures were grown from single, purified colonies at 37° C. overnight in L broth plus the appropriate antibiotic (example, kanamycin 50 μg/ml). Expression of the protein was determined by the pNB assay (See, Example 1) after lysis of the cells. Expression of the perhydrolase using the T7 expression system requires induction of the culture with the addition of IPTG (e.g., 100 mmole IPTG added at an OD₅₅₀ of 0.4). Overnight cultures, inoculated from a single colony, are used to inoculate the expression culture of the desired volume (25 mls to several liters) at an OD₅₅₀ of 0.1. The expression culture was then grown at the desired temperature (e.g., 25° C., 30° C., 37° C.) until an OD₅₅₀ of 0.4 was reached, after which IPTG was added. Expression was allowed to continue for 3 hours to overnight. Protein expression was monitored by pNB activity assay as described in Example 1. Usually, expression from the T7 system gives a high titer of protein, sufficient for further analysis such as crystallography.

Bacillus species are well-known as suitable hosts for expression of extracellular proteins (e.g., proteases). Intracellular expression of proteins is less well known. Expression of the perhydrolase protein intracellularly in Bacillus subtilis can be done using a variety of promoters, including, but not limited to pVeg, pSPAC, pAprE, or pAmyE in the absence of a signal sequence on the 5′ end of the gene. In some embodiments, expression is achieved from a replicating plasmid (high or low copy number), while in alternative embodiments, expression is achieved by integrating the desired construct into the chromosome. Integration can be done at any locus, including but not limited to the aprE, amyE, or pps locus. In some embodiments, the perhydrolase is expressed from one or more copies of the integrated construct. In alternative embodiments, multiple integrated copies are obtained by the integration of a construct capable of amplification (e.g., linked to an antibiotic cassette and flanked by direct repeat sequences), or by ligation of multiple copies and subsequent integration into the chromosome. In some embodiments, expression of the perhydrolase with either the replicating plasmid or the integrated construct is monitored using the pNB activity assay (described herein) in an appropriate culture.

As with Bacillus, in some embodiments, expression of the perhydrolase in the Gram-positive host Streptomyces is done using a replicating plasmid, while in other embodiments, expression of the perhydrolase is accomplished via integration of the vector into the Streptomyces chromosome. Any promoter capable of being recognized in Streptomyces finds use in driving transcription of the perhydrolase gene (e.g., glucose isomerase promoter, A4 promoter). Replicating plasmids, either shuttle vectors or Streptomyces only, also find use in the present invention for expression (e.g., pSECGT).

Structure of M. smegmatis Perhydrolase

The crystal structure of the M. smegmatis perhydrolase was determined to 2.2 Angstroms. The structure confirmed findings with gel filtration sizing columns, that indicated this enzyme is an octamer. The structure of the monomer places the enzyme in the class known as SGNH-hydrolases (See e.g., Molgaard et al., Structure 8: 373-383 [2000]). The active site residues were identified as S11-D192-H195, based on homology, confirming the identification of the catalytic triad based on loss of activity in the S11A, D192A, and H195A mutations described above. FIG. 3 provides schematics showing the structure of the M. smegmatis perhydrolase, as well as other serine hydrolases. As indicated, this enzyme has a different structure than the enzymes shown here (chymotrypsin, subtilisin, and α/β hydrolase). Indeed, the structural analysis of the perhydrolases of the present invention indicates that this group of enzymes has a different form and active site than do these other enzymes. A schematic diagram of the structure of the monomer is illustrated in FIG. 4. The structures of four other enzymes in the SGNH-hydrolase family have been solved, namely Aspergillus aculeates rhamnogalucturonan acetylesterase (RGAE), Bos taurus platelet activating factor (PAF-AH(1b)a), Streptomyces scabies esterase (SsEst) and the thioesterase/Protease I/Phospholipase L₁ (TAP or Tes) from E. coli. Very little sequence or functional homology is present in these enzymes. Basically, the sequence identity is reserved for the residues involved in the active site and those defining the family. While the overall folding of the enzymes is similar (See e.g., Molgaard et al., supra [2000], for overlaying of structures), there are structural differences. For example, there is a loop covering the active site in SsEst, compared to RGAE and TAP which have active sites that are surface-exposed. The M. smegmatis perhydrolase has an active site that is somewhat buried. The binding residues of the M. smegmatis perhydrolase were identified as Cys7, Asp10, Ser11, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194, and Phe196. These sites were derived from direct observation and by modeling studies to model substrate binding to the enzyme, using methods known in the art.

As indicated above, the M. smegmatis perhydrolase was found to be an octamer in the crystalline state. However, it is contemplated to be either a hexamer or octamer in solution. The octamer is seen to be a tetramer of dimers, two molecules are much more closely and extensively interacting and these are termed the “act transferase” dimers. Several of the conserved sites are found along this dimer interface. For example, residues Trp 14, Arg 27, Arg 56, His 81 and Pro 83, were found to be conserved in natural isolates that have perhydrolase activity and are contemplated to be critical in forming the interface. In addition one other residue, Glu 51, which is conserved in all but one of the natural isolates (and in that case it is a homologous enzyme) was identified.

One additional feature of interest in that in the natural isolates showing perhydrolase activity, all share an insertion of residues 69-81. This region forms a loop that is at the dimer interface. Without this loop, it is believed that much of the dimer interface would be lost and it is likely that dimers and subsequent aggregation would not occur. Thus, there is a correlation of the insertion with the structural aggregation particularly dimer formations and the appearance of perhydrolase activity. However, it is not intended that the present invention be limited to any particular mechanisms.

Key residues were found to be associated with desired activity in selected homologs. Indeed, there are several conserved residues that are contemplated to have importance for acyltransferase activity. These include Leu 6, Trp 14, Arg 27, Trp 34, Asp 62, Leu74, Leu 78 His 81, Pro83, Met 90, Lys 97, and Leu 114.

In additional analyses, the association of the perhydrolase with carbamate was investigated. The native octamer was determined in space group P4 with unit cell dimensions:

a=98.184 b=98.184 and c=230.119 α=90.00 β=90.00 γ=90.00, this crystal diffracted to about 2.0 Å. The carbamate-inhibited crystal grew in the space group P1 with unit cell dimensions a=67.754, b=80.096, and c=85.974 α=104.10°, β=112.10°, and γ=97.40° and these crystals diffract to a resolution exceeding 1.0 Å.

The carbamate was bound in a manner to exploit the interactions between the keto oxygen of the carbamate and residues forming the oxyanion hole, the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobic side chain extends along the hydrophobic surface of the binding site out into the surface opening between pairs of dimers in the octamer structure. The carbamate moiety direction highlights the pivotal role of the S54V mutation. The hydrophobic moiety passes adjacent to the side chain of ser 54. Mutating the serine side to valine increased the hydrophobicity, and also served as a gatekeeper to prevent hydrophilic nucleophiles (e.g., water) for competing with desired deacylating nucleophiles. The residues surrounding the carbamate moiety on the same and neighboring molecules forming the extended entry are expected to influence the selection of the optimal de-acylating nucleophile. The structure showed that each monomer was inhibited with carbamate covalently attached. Thus, all octamer active sites were found to be active and functional. The side chain of carbamate resembles the leaving groups of the substrates tested. Thus, the carbamate moiety indicates the access direction for substrate.

M. smegmatis Perhydrolase is an SGNH-Hydrolase

The perhydrolase of the present invention has certain components that indicate it is in the SGNH-hydrolase family of enzymes. This family is defined by having the four conserved amino acids SGN and H in four blocks, similar to the blocks that describe the lipolytic family of enzymes (See, Upton and Buckley, supra). In the case of the M. smegmatis perhydrolase, these correspond to S11, G52, N94 and H195 which correspond to Blocks III, III and V according to Upton and Buckley (Upton and Buckley, supra) and Molgaard et al. (Molgaard et al., supra). These amino acids are also conserved within the closest sequence homologs of the perhydrolase.

As indicated herein, the sequences were aligned using the Alignment program in Vector NTi (Informax, Invitrogen) In the following alignment providing a comparison of homolog sequences, the double underline indicates the residues involved in the active site. AR: Agrobacterium rhizogenes Q9 KWA6; RR: Rhizobium rhizogenes NF006; SM: Sinorhizobium meliloti RSM02162; MS: Mycobacterium smegmatis Act; MP: Mycobacterium parafortuitum Phd partial sequence; PD: Prosthecobacter dejongeii RVM04532. The amino acids within the blocks defining the SGNH-hydrolase family are indicated in bold letters.

                  Block I                                     Block II                     GDS                                         G AR(1) ----------MAESRSILCFGDSLTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED--PN RR(1) ----------MAESRSILCFGDSLTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED-PN RM(1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSP TLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDD-PN SM(1) -----------MVEKRSVLCFGDSRTWGWIPVKESSP TLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDD-PN MS(1) -------------MAKRILCFGDSLTWGWVPVEDGAP TERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDD-PT MP -------------GIRRILSFGDSLTWGWIPVEEGVP TERFPRDVRWTGVLADLLGDRYEVIEEGLSARTTTAED-PA PD(1) --------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTGVLAKALGAGFRVIEEGQNGRTTVHED--PL                             Block III                            GxND AR(67) DPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGKLAGQVLTSAGGIGTPYPAPKLLIVSPPPLAP RR(67) DPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGLKAGQVLTSAGGIGTPYPAPKLLIVSPPPLAP RM(78) DARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAP SM(67) DARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAP MS(65) DPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAP MP(65) DPRLNGSQYLPSCLASHLPLDLVILMLATNDTKANFGRTPFDIATGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGE PD(65) NICRKGKDYLPACLESHKPLDLVILMLGTNDLKSTFNVPPGEIAAGAGVLGRMILAGDAGP-ENRPPQLLLMCPPKVRDL                                               Block V                                                DGIHF (SEQ ID NO: 14) AR(147) MPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAA (SEQ ID NO: 15) RR(147) MPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAA (SEQ ID NO: 16) RM(158) MPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF-------- (SEQ ID NO: 17) SM(147) MPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF-------- (SEQ ID NO: 18) MS(145) MPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL-------- (SEQ ID NO: 19) MP 145) LPHPWFDLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDMVDGI------------------------------ (SEQ ID NO: 20) PD(144) SAMPDLDAKIPHGAARSAEFPRHYKAQAVALKCEYFNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG-------

The primers used to identify homologs for each of the Blocks indicated above are provided below:

Block I (forward 5′-3) 1e: acggtcctgtgctttggngaytcnyt (SEQ ID NO: 21) 1f: acggtcctgtgctttggngayagyyt (SEQ ID NO: 22) 1g: gcggtcctgttctwnggngaytcnyt (SEQ ID NO: 23) 1h: gcggtcctgttctwnggngayagyyt (SEQ ID NO: 24) 1i: gctcgaaccgtcctctgttttggngaytcnyt (SEQ ID NO: 25) 1j: gctcgaaccgtcctctgttttggngayagyyt (SEQ ID NO: 26) 1k: gctcgaaccgtcctctgtttnggngaytc (SEQ ID NO: 27) 1l: gctcgaaccgtcctctgttttggngaytcnytn (SEQ ID NO: 28) 1m: gctcgaaccgtcctctgttttggngaytcnytg (SEQ ID NO: 29) 1A: gccaagcgaattctgtgtttcggngaytcnyt (SEQ ID NO: 30) 1B: gccaagcgaattctgtgtttcggngayagyyt (SEQ ID NO: 31) Block III (reverse 5′-3) 3c: attccgcgcttcagrtcrttnvtncc (SEQ ID NO: 32) 3d: attccgcgcttcagrtcrttnwgncc (SEQ ID NO: 33) 3e: attccgcgcttcagrtcrttnscncc (SEQ ID NO: 34) 3f: attccgcgcttcagrtcrttnrancc (SEQ ID NO: 35) 3k: attccgcgcttcagrtcrttnrtncc (SEQ ID NO: 36) 3l: attccgcgcttcagrtcrttnytncc (SEQ ID NO: 37) 3m: attccgcgcttcagrtcrttnsgncc (SEQ ID NO: 38) 3n: attccgcgcttcagrtcrttnwcncc (SEQ ID NO: 39) 3o: attccgcgcttcagrtcrttnyancc (SEQ ID NO: 40) 3p: attccgcgcttgrsrtcrttnrtncc (SEQ ID NO: 41) 3q: attccgcgcttgrsrtcrttnytncc (SEQ ID NO: 42) 3r: attccgcgcttgrsrtcrttnsgncc (SEQ ID NO: 43) 3s: attccgcgcttgrsrtcrttnwcnnn (SEQ ID NO: 44) 3t: attccgcgcttgrsrtcrttnyancc (SEQ ID NO: 45) 3A: gcgccggaagtaggccttggtrtcrttnvtncc (SEQ ID NO: 46) 3B: gcgccggaagtaggccttggtrtcrttnwgncc (SEQ ID NO: 47) 3C: gcgccggaagtaggccttggtrtcrttnscncc (SEQ ID NO: 48) 3D: gcgccggaagtaggccttggtrtcrttnrancc (SEQ ID NO: 49) Block III (forward 5′-3) 3g: cggaattatcatgctgggnabnaayga (SEQ ID NO: 50) 3h: cggaattatcatgctgggncwnaayga (SEQ ID NO: 51) 3i: cggaattatcatgctgggngsnaayga (SEQ ID NO: 52) 3j: cggaattatcatgctgggntynaayga (SEQ ID NO: 53) 3u: ccggaattatcatgctnggnabnaayga (SEQ ID NO: 54) 3v: ccggaattatcatgctnggncwnaayga (SEQ ID NO: 55) 3w: ccggaattatcatgctnggngsnaayga (SEQ ID NO: 56) 3x: ccggaattatcatgctnggntynaayga (SEQ ID NO: 57) Block V (reverse 5′-3) 5c: acccttagcgtttggrtgnrtnccrtc (SEQ ID NO: 58) 5d: atccttagcgtttggrtgnavnccrtc (SEQ ID NO: 59) 5e: aatcttagccgtgrrrtgnrtnccrtc (SEQ ID NO: 60) 5f: aatcttagccgtgrrrtgnrcnccrtc (SEQ ID NO: 61) 5g: aatcttagccgtgrrrtgntrnccrtc (SEQ ID NO: 62) 5h: ccgctggtcctcatctggrtgnrtnccrtc (SEQ ID NO: 63) 5i: ccgctggtcctcatctggrtgnrcnccrtc (SEQ ID NO: 64) 5j: ccgctggtcctcatctggrtgntrnccrtc (SEQ ID NO: 65) 5k: ccgctggtcctcatcraartgnrtncc (SEQ ID NO: 66) 5A: cgattgttcgcctcgtgtgaartgnrtnccrtc (SEQ ID NO: 67) 5B: cgattgttcgcctcgtgtgaartgnrcnccrtc (SEQ ID NO: 68) 5C: cgattgttcgcctcgtgtgaartgntrnccrtc (SEQ ID NO: 69)

As described in greater detail herein, the sequence and structure results are supported by the activity data that indicate the perhydrolase enzymes of the present invention differ from lipolytic enzymes known in the art.

Identification of Homologs

As well known in the art, proteins with a desired activity may be identified in several ways, including but not limited to: 1) searching available databases for proteins with sequence homology (30-100%); 2) screening environmental isolates for the desired activity; and 3) examining type strains from ATCC of the genus identified to have activities (e.g., Mycobacterium and Corynebacterium, as described herein in particular embodiments).

By doing a standard protein-protein BLAST search, several homologs were identified from fully or partially sequenced genomes. From the known gene sequence, several homologs were amplified by PCR from the chromosome of the parent organism and cloned into a pET expression vector, essentially as described for the cloning of phd from M. smegmatis into pET16b. Homologues identified by this BLAST search included: Agrobacterium rhizogenes Q9 KWA6, A. rhizogenes Q9 KWB 1 A. tumefaciens Q8UFG4, A. tumefaciens Q8UAC0 (now AgrL, identical to 7-ACA arylesterase), A. tumefaciens Q9ZI09, A. tumefaciens (radiobacter)ACA, Prosthecobacter. dejongeii RVM04532, Rhizobium. loti Q98MY5, R. meliloti Q92XZ1, R. meliloti Q9EV56, R. rhizogenes NF006, R. rhizogenes NF00602875, R. solanacerarum Q8XQI0, Sinorhizobium meliloti RSM02162, S. meliloti RSM05666, Mesorhizobium loti RML000301, A. rhizogenes Q9 KWA6, and A. rhizogenes Q9 KWB1.

Based on these results, a homology tree of proteins with sequence homology (20-80%) to M. smegmatis perhydrolase was generated. As shown in FIG. 2, an enzyme in the family of lipolytic enzymes described by Upton and Buckley (supra) is that of V. mimicus. This phylogenetic tree was generated using the alignment program in Vector NTi (Informax, Invitrogen). The green arrow indicates M. smegmatis perhydrolase, the red arrow indicates A. radiobacter 7-ACA arylesterase, the blue arrow indicates E. coli TAP, and the black arrow indicates A. aculeates RGAE.

As further indicated in FIG. 2, the perhydrolase is not closely related to this enzyme. The perhydrolase and its closest relatives, Prosthecobacter dejongeii RVM04532, R. rhizogenes NF006, A. rhizogenes Q9 KWA6, R. meliloti Q92XZ1, S. meliloti RSM02162, A. rhizogenes Q9 KWB1 and R. rhizogenes NF00602875 come off their own branch (i.e., a branch that is different from the 7-ACA arylesterase-like proteins and the RGAE/TAP-like proteins). However, it is contemplated that some additional, more distantly related homologs will find use in the present invention due to perhydrolase activity or will serve as a suitable backbone for modification to the desired perhydrolase activity.

In addition to the sequence and homology analysis, environmental isolates were grown on a rich medium (N-MISO: g/l: glucose 10 g, yeast extract 10 g, KNO₃ 1.5, KH₂PO₄ 3.4 g, NaH₂PO4.H₂O 3.4 g, Salt Solution C 10 ml [Salt Solution C: g/l: MgSO₄.7H₂O 25, FeSO₄7H₂O 2.8, MnSO₄H₂O1.7, NaCl 0.6, NaMoSO₄2H₂O, ZnSO₄.7H₂O 0.06, in 0.1N HCl]), assayed and those positive for the transesterification reaction were purified as described in the Examples. This is one of the screening methods that can be used to identify perhydrolase. These data show that the present invention finds use in identification of additional enzymes with the desired perhydrolase activity.

Additional Investigations of Homologues

In addition to the above analyses, an enzyme library of novel “GDSL-type” esterases which are homologous to the prototype M. smegmatis perhydrolase was created. In order to identify new “GDSL”-type esterases, a sequence homology based screening procedure was established and used to screen libraries set up from complex metagenomic DNA (at BRAIN). An enzyme library comprising 19 “GDSL”-type esterases (See, below) was developed. The sequences in this library were:

S248_M2bB11 (DNA) (SEQ ID NO: 70) ATGTTCGCGCTTTGCACGGCCGCGTCAGCGGCCCCCGATCGCACCGTCGT CTTTTTTGGGGACAGCCTGACCGCGGGGTACGGCCTCGATGACCCGCAGA CCCAGTCCTACCCGGCCAGGATCCAGGAGAAGGTCGACGCCGCGGGCCTG CGCTGGAAGGTCGTGAATGCCGGCCTCTCGGGCGAGACGAGCGCCGGCGG CCTGCGGCGGGTCGACTGGGTGCTCGGCCAGCACATCGACGCCTTTGTCC TGGCGCTTGGCGCCAACGATGGCCTGCGGGGGATCGACCCCCAGGTCACG AGGGCCAATCTCCAGGAGATCATCAACCGGGTCCGCTCCCGGTGGCCCCG CGCGGCGATCGTCATCGCCGGGATGAAAATGCCCCAGAGCATGGGACAGG ACTACGCCGCGAATTTTGACCGGATCTTCCCCGGTCTCGCCGCGAGGAAT TCGGCCACGCTCATCCCCTTTCTATTAGAAGGGGTCGCCGCCCATCCTAG CCTCAACCAAGGCGACGGCATCCACCCGACGGCCGCCGGGGACGCACTCG TTGCAGGGACCGTGTGGACGTACCTGCTTCCGATCCTGCGGTCAGCACAC TAA S248_M2bB11 (Amino Acid) (SEQ ID NO: 71) MFALCTAASAAPDRTVVFFGDSLTAGYGLDDPQTQSYPARIQEKVDAAGL RWKVVNAGLSGETSAGGLRRVDWVLGQHIDAFVLALGANDGLRGIDPQVT RANLQEIINRVRSRWPRAAIVIAGMKMPQSMGQDYAANFDRIFPGLAARN SATLIPFLLEGVAAHPSLNQGDGIHPTAAGDALVAGTVWTYLLPILRSAH S248_M40cD4 (DNA) (SEQ ID NO: 72) ATGCGCTTTGCTAAGCTCACTGCCGTCATCTTTGCCCTGATAGTCTTGCA CAGCCCCCTTGCCGCCGCCGCGCCGCCCACCGTGATGGTGTTTGGCGACA GTCTGACCGCCGGGTTGGGATTGCCGGCCGATGCTGCATTTCCGGCGCAG CTCCAGGCAAAGCTGCACGATATGGGTATCCTGCAGAAATCGCCGCGCGC GCCACCTCGGGGCAAACGACGGCCGGCGGGTTGGCGAGCCTTGCGGATGC GCTGGCCGCAAAGCCGGATTTGGTGATCCTCGAACTCGGCGCCAATGACA TGCTGCGCGCGGTCGATCCGGCCAGCGTGCGCGCCAATCTCGATGCAATG ATGACGAAAATCCAGGCGAGCGGCGCTAAACTGCTGCTGACCGGAATGCA GGCGGCGCCCAATTGGGGCGAGGACTATAAGCACGATTTCGACCGCCTTT ATCCCGAGCTTGCGAAGGCGCACGGGGTGACGCTTTATCCATTCTTTCTT GATGGGGTGGCGCTGGACCCGGCGCTGAACCAGGCGGATGGAATGCACCC GAACGCCAAGGGGGTCGCCGTGATCGTCGACCGTATCGCGCCCGTCGTCG CCAAGATGCTGAGAGGCCAGTCATAA S248_M40cD4 (Amino Acid) (SEQ ID NO: 73) MRFAKLTAVIFALIVLHSPLAAAAPPTVMVFGDSLTAGLGLPADAAFPAQ LQAKLHDMGIPAEIAARATSGQTTAGGLASLADALAAKPDLVILELGAND MLRAVDPASVRANLDAMMTKIQASGAKLLLTGMQAAPNWGEDYKHDFDRL YPELAKAHGVTLYPFFLDGVALDPALNQADGMHPNAKGVAVIVDRIAPVV AKMLRGQS S248_M44aA5 (DNA) (SEQ ID NO: 74) ATGATCGCATGGCTTACCGGATGCGGCAGCGCAAAGACGCAACCGCAGCC CGCAAGTTCCATCCCGCCATCCAGTATTCCAGCAACCGCAAAACCTGCGA CAACGGATATCAGACCGATCATCGTTGCTTTCGGCGACAGCCTGACTGCA GGATACGGCGTCAGTAGTGAACAAAGCTATCCGGCCAATCTTCAACGCGA TCTGGATGCGCGTGGATATCATGCCCACGTCATCAACGAAGGCATCAGCG GCAACACATCGAAAGACGGCGTTCTCAGGGCCCAGGCGATTGCGGCACTC CATCCGGCTGTCGTCATCGTTGCCTTCGGCGGCAACGACGGTCTGCGTGG CCTCCCCATCGGAGACACGGAAATGAATCTGGCAACGATCATCTCAACCA TGCAGCATGCCCATGCCAAGGTAATTTTAGGCGGAATTACTTTGCCTCCC AACTATGGCAGCGAATACATCGCCAAATTCAATGCGATCTATAAAAAGCA GGCAGCCGCGTATCATGTGCCCCTGCTGCCCTTCATGCTGAAGGGGGTGT ATGGCGTGCCCGGTTCCATGCAGAGCGACGGCATCCATCCGACCGCCAAG GGCTGCCAGCAAGTGGCCAGAAACTTCCTGCCCTTGTTATTGCCGCTCCT GCACAAATCAGGGAAGAAATCCATGGAGTCGAAAGCATTGTCTCGACGTC ATTAA S248_M44aA5 (Amino Acid) (SEQ ID NO: 75) MIAWLTGCGSAKTQPQPASSIPPSSIPATAKPATTDIRPIIVAFGDSLTA GYGVSSEQSYPANLQRDLDARGYHAHVINEGISGNTSKDGVLRAQAIAAL HPAVVIVAFGGNDGLRGLPIGDTEMNLATIISTMQHAHAKVILGGITLPP NYGSEYIAKFNAIYKKQAAAYHVPLLPFMLKGVYGVPGSMQSDGIHPTAK GCQQVARNFLPLLLPLLHKSGKKSMESKALSRRH S261_M2aA12 (DNA) (SEQ ID NO: 76) ATGAAAAACATCCTTGCATTTGGCGACAGTCTGACCTGGGGTTTTGTGGC CGGACAGGATGCGCGCCATCCGTTTGAAACCCGCTGGCCAAACGCATTGG CGGCCGGCCTTGGGGGCAAAGCCCGCGTAATTGAAGAGGGTCAGAACGGC CGCACTACGGTGTTCGACGATGCCGCCACCTTCGAATCTCGAAATGGCTC GGTGGCATTGCCGCTGCTACTGATCAGCCACCAGCCGTTGGACCTGGTAA TCATCATGCTCGGCACCAATGACATCAAGTTTGCCGCCCGCTGCCGCGCC TTTGATGCTTCAATGGGCATGGAACGGCTGATCCAGATCGTCAGAAGTGC CAACTACATGAAGGGCTACAAGATACCTGAAATCCTCATCATATCGCCGC CCAGCCTCGTGCCGACGCAGGATGAATGGTTCAACGACCTCTGGGGCCAT GCCATCGCCGAGTCAAAACTCTTCGCCAAGCACTACAAGCGCGTGGCCGA AGAACTGAAAGTGCATTTCTTTGATGCAGGCACGGTGGCCGTCGCCGACA AGACCGACGGCGGACATCTCGATGCTGTGAATACTAAAGCCATTGGCGTC GCATTGGTGCCGGTGGTGAAATCAATACTCGCTCTCTAA S261_M2aA12 (Amino Acid) (SEQ ID NO: 77) MKNILAFGDSLTWGFVAGQDARHPFETRWPNALAAGLGGKARVIEEGQNG RTTVFDDAATFESRNGSVALPLLLISHQPLDLVIIMLGTNDIKFAARCRA FDASMGMERLIQIVRSANYMKGYKIPEILIISPPSLVPTQDEWFNDLWGH AIAESKLFAKHYKRVAEELKVHFFDAGTVAVADKTDGGHLDAVNTKAIGV ALVPVVKSILAL S279_M70aE8 (DNA) (SEQ ID NO: 78) ATGCCGAAAATAGCCAAACTCGCGCCGTCGGATGTGATCGTAGCTTTCGG CGACAGTCTGACGTTCGGCACCGGCGCAACGGAAGCGGAGAGTTATCCCA TCGTGCTCGCACAATTGATCGGTCGCACCGTGGTGCGCGCGGGTGTGCCG GGTGAGGTAACCGAAGGCGGGCTTGCGCGCCTGACCGACGTTATCGAAGA ACACAAGCCGAAGCTGATTATTGTTTGCCTGGGCGGCAACGACATGCTGC GCAAGGTCCAGGAAGACCAGACCCGCGCCAATTTGCGCGCCATTATTAAA ACCATCAAGGCGCAAGGCATCGCCGTGGTACTGGTCGGTGTGCCGAAGCC CGCGCTGGTGACCAGTGCGCCGCCGTTCTACGAGGAGATCGCCAAAGAGT TCGGTATCCCTTACGAAGGCAAGATTGTTACCGACGTGTTGTACCAACGC GATCAGAAATCCGATTCCATACATCCCAATGCCAAAGGCTATCGGCGCAT GGCCGAAGCGATAGCCACGCTGCTGAAAAAATCCGGAGCCATTTAA S279:M70aE8 (Amino Acid) (SEQ ID NO: 79) MPKIAKLAPSDVIVAFGDSLTFGTGATEAESYPIVLAQLIGRTVVRAGVP GEVTEGGLARLTDVIEEHKPKLIIVCLGGNDMLRKVQEDQTRANLRAIIK TIKAQGIAVVLVGVPKPALVTSAPPEYEEIAKEFGIPYEGKIVTDVLYQR DQKSDSIHPNAKGYRRMAEAIATLLKKSGAI S279_M75bA2 (DNA) (SEQ ID NO: 80) ATGGAACGGACCGGCCGCGCTGGCGATCGGTGTCGGCGTGGGGCTGGCGA GCCTGAGCCCGGTCGCGCTGGCGACGCCGCCGCGGGGCACCGTGCCGGTG TTCACCCGATCGGGGACAGCCTGACGGACGAGTATTTTGAGCCGTTCTTC CAGTGGGGGTTCTGCGGGAAGTCGTGGGCCGAGATTTTGGTGGAGACGGG GCGGGCGAGCATGGGCCCGACGGCGCAGCAGGCGGGGATCAGCGAGCCGG AGGGATGGTCGGATCCGCGGAACACGGGGTATCAGCACAACTGGGCGCGG TACTCGTGGAGCTCCTCAGACGCGCTGACCGAGGAGTCGCCGGGGGCGAC GCTGAGCGTGCTGCTTGGGGCGGAGTACGCGGTGGTGTTCATTGGGACCA ACGACTTCAATCCGTCGTGGCCGGCGTATCAGAGCGTGTATCTGAGCCAG TGGAGCGACGAGCAGATCGACACGTACGTGAACGGGGTGGTGCAGAACAT CGCGCAGATGGTGGACTCGCTGAAGTCGGTCGGGGCGAAGGTGGTGCTTG CGCCGCCGGTGGATTTTCAGTTCGCGGGGTTCCTGCGGAACTCATGCCCG GATCCGATGCTGCGCGAGCAGGCGGGTATTCTGACACGGAAGTGCCACGA CCGGGTGCGGTCGATGGCGCGGCAGAAGCACGTGGTGTTCGTGGACATGT GGCGGCTGAACCGCGATTTGTTCGGCAACGGGTTCGCGATCAGCTACGGC CTTCGGAACACGGTGCGCGTGGGGGACTCGGAGATCGGGCTGCAACTGGC CGGGCTGACGGGATCGGCGGGGCTGGTTCCGGACGGGATCCATCCGCAGC GGGTGGTGCAGGGGATCTGGGCGAATGCGTTCATCGTGGGTCTGAACGCG CATGGGGCGAACATCGCGCCCATCGGCGAGGCGGAGATGTGCGCGATGGG GGGGGTCGTGTACGGGGGAACGGACACGCTGGCGAACTTCCTGCCGCCGG TCGCGGGCTACGTGGAGGACTTCCGCAACGCGGGGGACTTCGTGTGCACG GCGGACTTCAACCATGACCTTGGCGTGACGCCGACGGACATCTTCGCGTT CATCAACGCGTGGTTCATGAATGATCCCTCGGCGCGGATGAGCAACCCGG AGCACACGCAGATCGAGGACATCTTCGTGTTTCTGAATCTGTGGCTGGTG GGGTGCTAA S279_M75bA2 (Amino Acid) (SEQ ID NO: 81) MERTGRAGDRCRRGAGEPEPGRAGDAAAGHRAGVHPIGDSLTDEYFEPFF QWGFCGKSWAEILVETGRASMGPTAQQAGISEPEGWSDPRNTGYQHNWAR YSWSSSDALTEESPGATLSVLLGAEYAVVFIGTNDFNPSWPAYQSVYLSQ WSDEQIDTYVNGVVQNIAQMVDSLKSVGAKVVLAPPVDFQFAGFLRNSCP DPMLREQAGILTRKCHDRVRSMARQKHVVFVDMWRLNRDLFGNGFAISYG LRNTVRVGDSEIGLQLAGLTGSAGLVPDGIHPQRVVQGIWANAFIVGLNA HGANIAPIGEAEMCAMGGVVYGGTDTLANFLPPVAGYVEDFRNAGDFVCT ADFNHDLGVTPTDIFAFINAWFMNDPSARMSNPEHTQIEDIFVFLNLWLV GC M091_M4aE11 (DNA) (SEQ ID NO: 82) ATGAAGACCATTCTCGCCTATGGCGACAGCCTGACCTATGGGGCCAACCC GATCCCGGGCGGGCCGCGGCATGCCTATGAGGATCGCTGGCCCACGGCGC TGGAGCAGGGGCTGGGCGGCAAGGCGCGGGTGATTGCCGAGGGGCTGGGT GGTCGCACCACGGTGCATGACGACTGGTTTGCGAATGCGGACAGGAACGG TGCGCGGGTGCTGCCGACGCTGCTCGAGAGCCATTCGCCGCTCGACCTGA TCGTCATCATGCTCGGCACCAACGACATCAAGCCGCATCACGGGCGGACG GCCGGCGAGGCCGGGCGGGGCATGGCGCGGCTGGTGCAGATCATCCGCGG GCACTATGCCGGCCGCATGCAGGACGAGCCGCAGATCATCCTCGTGTCGC CGCCGCCGATCATCCTCGGCGACTGGGCGGACATGATGGACCATTTCGGC CCGCACGAAGCGATCGCCACCTCGGTGGATTTCGCTCGCGAGTACAAGAA GCGGGCCGACGAGCAGAAGGTGCATTTCTTCGACGCCGGCACGGTGGCGA CGACCAGCAAGGCCGATGGCATCCACCTCGACCCGGCCAATACGCGCGCC ATCGGGGCAGGGCTGGTGCCGCTGGTGAAGCAGGTGCTCGGCCTGTAA M091_M4aE11 (Amino Acid) (SEQ ID NO: 83) MKTILAYGDSLTYGANPIPGGPRHAYEDRWPTALEQGLGGKARVIAEGLG GRTTVHDDWFANADRNGARVLPTLLESHSPLDLIVIMLGTNDIKPHHGRT AGEAGRGMARLVQIIRGHYAGRMQDEPQIILVSPPPIILGDWADMMDHFG PHEAIATSVDFAREYKKRADEQKVHFFDAGTVATTSKADGIHLDPANTRA IGAGLVPLVKQVLGL Est105 (DNA) (SEQ ID NO: 84) ATGCGCACGCTTCACCGAAGCCTGCTCGCAAGCGCGGCCGCGCTTTTTCT AGCGGCATCCGGCAACGCAACGGCGCAGTTCTCGAACGTCTATTTCTTCG GCGACAGCCTGACCGACGCGGGTTCCTTCAAGCCTGTGCTGCCTCCTGGT ACAGGATTATTCACGACGAATCCCGGCCCGGTATGGCCGCAGGTATTCGG GGCGAACTACGGCGTCGCGGTGACGCCCGCAAACCAGGGTGGGACCGATT ATGCGCAGGGTGGCGCGCGCGTGACGAGCCTGCCTGGCGTTCCGACGTCG CAGCCGACCGGCAGCGCGGTACCGATCGCTACGCAGATTTCGCAGTTCCT CGGCTCGGGTCCGGCGGATCCGAACGCATTCTATTCGGTGTGGGGCGGCG CGAACGACATCTTTTTCCAGCTGGGGTTGGCGCAGGCGGGCATGGCGACG CCGGCGCAGGTCCAGTCGGCCGTCGGCTTGGCCGCGGTCCAGCTGGCGCA GGCAACTGCGGCGCTCAACGCCAGCGGCGCGCGATTCATCACGGTTATCA ACGTGCCGGACATCGGTAAAACGCCGTTCGGCGTCGGCTCCGGTCAAGGA GCGCAGATCACCGCTCTGTCGTCTTTCTTCAACAGCACGCTGTTCGGCGC GCTCGACGCCACGGGCATCCAGACGATGCGCGTGAACGGGTTCGCGGTGC TGAACGAGGTGGTCGCGGACCCGGCGGCTTATGGCTTCGCGAATGCATCA ACGCCAGCGTGCGGGGCCACGCCATCGCTCGTCTGCACGTCGGCGAACTT CGTCACGCCCTTGGCCGCGCAGACCTTCCTCTTCGCAGACGGCGTTCACC CCACCACGGCCGGGCACGCCCTCATCGCCCAAGCGGTCCAGGCGATGATC ACCGGTCCCCAACAGATGGCGGCGTTGGGCGACGCCCCGCTCGCCGTCGA GCAGGCCAACTTCCGCGCGCTCGACAACCGCATGTGGTCGAGCCTCAATG CGCCGCGCAGCCCGGGCAAGCTCCAGGGTTGGGCGGCCTACGACTACAGC CACACGGACCTGCAGGCGGGACCGACCAATGGCAGCGGACACATGAACAC CGTTGCGGTCGGGGTCGACATGAAAGTCTCCGATCATATGCTCGCCGGCG CGATGTTCGGCTATACCAACACCAAGGGCGACTTCGGCGGCCCCGGCGGC GGATACACACTGAAGCAGCCTGTGGGCACTGCCTATGCGGGTTACGGCGT GGGCCCTTGGTATGTCGGCGCGACGCTCGGCACAGGTGGCCTCGACTACT CGGACGTCACGCGCGCCATCCCGCTTGGCTTGGCGGTTCGCACCGAGAGC GCCGAGGCCCGAGGCTACGAGTTCACGGGCCGGATCCTCGGCGGCTACTG GTTCACGATGCGCGACCTGATGCACGGGCCGTACGCGCGTCTCGCGTGGA CGAAGGCCGTCGTCAAGCGGTTTTCCGAGGAGAGCACCGACAGCACGGCG TTGAACTACGACAGGCAGGAGCGCAAGCAACTGCTGTGGAGCCTCGGATG GCAACTCGCCGGCAACGTCGGCAGCATCCGTCCCTACGCGCGGGCGACCT GGGAGATCGACTCCAAGGATCAGGACCGCAGCGTTGGCGCATCGTCGGTC ACGCTGGGCGGCTTTTACAGTGTTCCGGTCGCGAAGCCGGACAATAGCTA TGCGCTCTTCAGCCTCGGCGCGAGTACCGAGCTCGGGAGCGTCACCGGGT TTGTCGCGGGCTCGGCCACCGCAGGCCGGGCGGATGCCAACTATTGGGCG GTCACGGTCGGCCTGCGGATGCCGTTGTAG Est105 (Amino Acid) (SEQ ID NO: 85) MRTLHRSLLASAAALFLAASGNATAQFSNVYFFGDSLTDAGSFKPVLPPG TGLFTTNPGPVWPQVFGANYGVAVTPANQGGTDYAQGGARVTSLPGVPTS QPTGSAVPIATQISQFLGSGPADPNAFYSVWGGANDIFFQLGLAQAGMAT PAQVQSAVGLAAVQLAQATAALNASGARFITVINVPDIGKTPFGVGSGQG AQITALSSFFNSTLFGALDATGIQTMRVNGFAVLNEVVADPAAYGFANAS TPACGATPSLVCTSANFVTPLAAQTFLFADGVHPTTAGHALIAQAVQAMI TGPQQMAALGDAPLAVEQANFRALDNRMWSSLNAPRSPGKLQGWAAYDYS HTDLQAGPTNGSGHMNTVAVGVDMKVSDHMLAGAMFGYTNTKGDFGGPGG GYTLKQPVGTAYAGYGVGPWYVGATLGTGGLDYSDVTRAIPLGLAVRTES AEARGYEFTGRILGGYWFTMRDLMHGPYARLAWTKAVVKRFSEESTDSTA LNYDRQERKQLLWSLGWQLAGNVGSIRPYARATWEIDSKDQDRSVGASSV TLGGFYSVPVAKPDNSYALFSLGASTELGSVTGFVAGSATAGRADANYWA VTVGLRMPL Est114 (DNA) (SEQ ID NO: 86) ATGGGGCGATCGAGAGTTCTGAAGGCTGTTTTCCTGGTGGCGTGCCTTGT GGGTCGGCTCGCGGCGCATGCCGAGGCGTCGCCCATCGTGGTCTACGGCG ATAGCCTCTCTGACAACGGCAATCTGTTTGCGCTCACCGGCGGTGTCGCG CCGCCCTCGCCGCCGTACTTCAACGGACGGTTTTCTAATGGCCCGGTGGC CGTGGAGTATCTCGCGGCCGCGCTGGGATCTCCGCTGATCGATTTCGCGG TCGGCGGGGCGACGACCGGCCTCGGCGTCAACGGCGATCCCGGTGGTTCG CCGACGAGTCTCGGCGCGGCGGGATTGCCGGGGCTTCAGACGACATTCGC CGCCACGCAAGGCACGCTGGGTCCGTACGTTGGTGGTCTCTTCGTGGTGT GGGCGGGTCCGAACGACTTCTTGTCGCCCTCGCCGCTTGACACGAACGCT TTTCAGATTGCGAACCGGGCCGTGTCCAACATCCTCGGCGTGGTGGCATC ACTTCAGGCACTCGGCGTCGAGCGCATCCTCGTCCCCGGCATGCCCGATC TCGGTCTGACGCCCGCTCTTCAGCCCATCGCAGGCGCAGCCACCGCGTTC ACCGATTTGTTCAACTCGATGCTGCGCGCGGGCTTGCCGAACGACGTGCT GTACCTGGACACGGCGACAATCTTCCGATCGATCGTGGCAGACCCTGGGG CCTACGGCTTGACCAACGTGACCACGCCGTGCCTGATTGGTGCGACCGTC TGCGCGAATCCGGATCAGTACCTGTTCTGGGATGGTATTCATCCTACGAC GGCGGGGCACGCGATCTTGGGCAATGCCCTCGTCGCCCAGGCAGTCCCCG AGCCCGCGACCATGGTGCTCGTGCTGACGGGTCTGTCCATGCACGTGATT GCGCGCCGGCGGCGGGCGTAA Est114 (Amino Acid) (SEQ ID NO: 87) MGRSRVLKAVFLVACLVGRLAAHAEASPIVVYGDSLSDNGNLFALTGGVA PPSPPYFNGRFSNGPVAVEYLAAALGSPLIDFAVGGATTGLGVNGDPGGS PTSLGAAGLPGLQTTFAATQGTLGPYVGGLFVVWAGPNDFLSPSPLDTNA FQIANRAVSNILGVVASLQALGVERILVPGMPDLGLTPALQPIAGAATAF TDLFNSMLRAGLPNDVLYLDTATIFRSIVADPGAYGLTNVTTPCLIGATV CANPDQYLFWDGIHPTTAGHAILGNALVAQAVPEPATMVLVLTGLSMHVI ARRRRA Sinorhizobium meliloti SmeI (SMa1993) (DNA) (SEQ ID NO: 88) ATGACAATCAACAGCCATTCATGGAGGACGTTAATGGTGGAAAAGCGCTC AGTACTGTGCTTTGGGGATTCGCTGACATGGGGCTGGATTCCGGTGAAGG GATCCTCACCGACCTTGCGCTATCCCTATGAACAACGGTGGACCGGCGCA ATGGCCGCGAGGCTTGGCGACGGTTACCACATCATCGAAGAGGGGCTGAG CGCCCGCACCACCAGCCTCGACGACCCCAACGACGCGCGGCTCAACGGCA GCACCTACCTGCCCATGGCACTCGCCAGCCACCTCCCACTCGACCTCGTC ATCATCATGCTGGGCACGAACGACACGAAATCCTATTTCCACCGCACGCC TTACGAGATCGCCAACGGCATGGGCAAGCTAGTCGGCCAGGTGCTGACCT GCGCCGGTGGCGTCGGCACGCCATATCCCGCGCCGAAGGTGCTTGTCGTC GCTCCGCCGCCGCTCGCGCCGATGCCCGACCCGTGGTTCGAAGGCATGTT CGGCGGCGGCTACGAGAAGTCGAAGGAACTCTCCGGCCTCTACAAGGCGC TTGCCGATTTCATGAAGGTCGAGTTTTTCGCCGCCGGTGATTGCATTTCC ACCGATGGGATCGACGGCATTCACCTCTCGGCGGAAACCAACATCAGACT CGGGCACGCGATCGCGGACAAAGTTGCGGCGTTGTTC Sinorhizobium meliloti SmeI (SMa1993) (Amino Acid) (SEQ ID NO: 89) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKGSSPTLRYPYEQRWTGA MAARLGDGYHIIEEGLSARTTSLDDPNDARLNGSTYLPMALASHLPLDLV IIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVV APPPLAPMPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCIS TDGIDGIHLSAETNIRLGHAIADKVAALF Sinorhizobium meliloti SmeII (Q92XZ1) (DNA) (SEQ ID NO: 90) ATGGAGGAGACAGTGGCACGGACCGTTCTATGCTTCGGAGATTCCAACAC TCACGGCCAGGTACCTGGCCGCGGACCGCTTGATCGCTACCGACGCGAAC AGCGCTGGGGCGGTGTTCTGCAAGGCCTGCTCGGCCCGAACTGGCAGGTT ATCGAAGAAGGCCTGAGCGGACGCACGACCGTGCATGACGATCCGATCGA AGGTTCGCTCAAGAACGGCCGGACCTATCTGCGCCCCTGTCTGCAGAGCC ATGCACCACTCGACCTTATCATCATTATGCTCGGCACCAATGACCTGAAG CGGCGCTTCAACATGCCACCGTCCGAGGTCGCAATGGGCATCGGCTGTCT CGTGCACGATATCCGAGAACTCTCGCCCGGCCGGACCGGCAACGATCCCG AAATCATGATCGTCGCCCCGCCGCCGATGCTGGAAGATCTCAAGGAATGG GAGTCGATTTTCTCAGGCGCACAGGAAAAATCTCGCAAGCTGGCGCTGGA GTTCGAGATAATGGCGGATTCTCTGGAGGCGCATTTCTTCGACGCCGGTA CGGTCTGCCAGTGTTCGCCGGCCGATGGCTTCCACATCGACGAGGATGCC CACCGCCTGCTCGGCGAGGCTCTCGCCCAGGAAGTGCTGGCGATCGGGTG GCCCGATGCGTAA Sinorhizobium meliloti SmeII (Q92XZ1) (Amino Acid) (SEQ ID NO: 91) MEETVARTVLCFGDSNTHGQVPGRGPLDRYRREQRWGGVLQGLLGPNWQV IEEGLSGRTTVHDDPIEGSLKNGRTYLRPCLQSHAPLDLIIIMLGTNDLK RRFNMPPSEVAMGIGCLVHDIRELSPGRTGNDPEIMIVAPPPMLEDLKEW ESIFSGAQEKSRKLALEFEIMADSLEAHFFDAGTVCQCSPADGFHIDEDA HRLLGEALAQEVLAIGWPDA Sinorhizobium meliloti SmeIII (Q9EV56) (DNA) (SEQ ID NO: 92) ATGAAGACAGTCCTTTGCTACGGTGACAGTCTGACCTGGGGATACGATGC AACCGGTTCCGGCCGGCATGCGCTGGAGGACCGTTGGCCGAGCGTGCTGC AGAAGGCGCTCGGTTCGGACGCGCATGTCATCGCCGAAGGGCTGAACGGG CGGACGACCGCCTATGACGACCATCTCGCCGATTGCGACCGGAACGGCGC GCGTGTCCTCCCGACGGTCCTGCACACCCACGCGCCACTCGATCTCATCG TGTTCATGCTCGGCTCGAACGACATGAAGCCGATCATTCACGGCACCGCT TTCGGCGCGGTGAAGGGCATCGAGCGCCTCGTCAATCTGGTGCGCAGGCA CGACTGGCCGACGGAAACGGAGGAGGGGCCCGAGATTCTCATCGTCTCGC CGCCGCCGCTCTGCGAGACGGCCAACAGCGCCTTTGCCGCCATGTTCGCG GGCGGGGTCGAGCAATCCGCAATGCTGGCGCCGCTTTATCGCGATCTCGC CGACGAGCTCGACTGCGGCTTCTTCGACGGCGGATCGGTGGCCAGGACGA CGCCGATCGACGGTGTCCACCTCGACGCGGAGAACACCCGGGCGGTCGGC AGAGGGTTGGAGCCTGTCGTGCGGATGATGCTCGGGCTTTAA Sinorhizobium meliloti SmeIII (Q9EV56) (Amino Acid) (SEQ ID NO: 93) MKTVLCYGDSLTWGYDATGSGRHALEDRWPSVLQKALGSDAHVIAEGLNG RTTAYDDHLADCDRNGARVLPTVLHTHAPLDLIVFMLGSNDMKPIIHGTA FGAVKGIERLVNLVRRHDWPTETEEGPEILIVSPPPLCETANSAFAAMFA GGVEQSAMLAPLYRDLADELDCGFFDGGSVARTTPIDGVHLDAENTRAVG RGLEPVVRMMLGL Agrobacterium tumefaciens Atu III (AAD02335) (DNA) (SEQ ID NO: 94) ATGGTGAAGTCGGTCCTCTGCTTTGGCGATTCCCTCACCTGGGGATCAAA TGCGGAAACGGGTGGCCGGCACAGCCATGACGATCTTTGGCCGAGCGTCT TGCAGAAGGCGCTCGGTCCTGACGTGCATGTGATTCACGAAGGTCTGGGT GGTCGCACCACCGCCTATGACGACAACACCGCCGATTGCGACCGCAACGG CGCGCGGGTTCTTCCGACGTTGTTGCACAGCCATGCGCCGCTGGATCTGG TGATTGTCATGCTCGGGACCAACGACCTGAAGCCGTCAATCCATGGATCG GCGATCGTTGCCATGAAGGGTGTCGAAAGGCTGGTGAAGCTCACGCGCAA CCACATCTGGCAGGTGCCGGACTGGGAGGCGCCTGACGTGCTGATCGTCG CACCGCCGCAGCTGTGTGAAACGGCCAATCCGTTCATGGGCGCGATCTTT CGTGATGCGATCGATGAATCGGCGATGCTGGCGTCCGTTTACCGGGACCT TGCCGACGAGCTTGATTGCGGCTTTTTCGATGCGGGTTCCGTCGCCCGAA CGACGCCGGTGGATGGCGTTCATCTCGATGCTGAAAATACGCGGGCCATC GGGCGGGGGCTGGAGCCCGTCGTTCGCATGATGCTCGGACTTTAA Agrobacterium tumefaciens Atu III (AAD02335) (Amino Acid) (SEQ ID NO: 95) MVKSVLCFGDSLTWGSNAETGGRHSHDDLWPSVLQKALGPDVHVIHEGLG GRTTAYDDNTADCDRNGARVLPTLLHSHAPLDLVIVMLGTNDLKPSIHGS AIVAMKGVERLVKLTRNHIWQVPDWEAPDVLIVAPPQLCETANPFMGAIF RDAIDESAMLASVYRDLADELDCGFFDAGSVARTTPVDGVHLDAENTRAI GRGLEPVVRMMLGL Mesorhizobium loti Mlo I (Q98MY5) (DNA) (SEQ ID NO: 96) ATGAAGACGGTGCTTTGCTACGGCGACTCGCTGACCTGGGGCTACAATGC CGAAGGCGGCCGCCATGCGCTGGAAGACCGCTGGCCGAGCGTGCTGCAAG CAGCGTTAGGCGCCGGCGTGCAAGTGATTGCCGATGGCCTCAACGGCCGC ACCACGGCCTTCGACGATCATCTGGCCGGTGCTGATCGCAACGGCGCCAG GCTGCTGCCGACGGTCCTGACGACGCACGCGCCGATCGACCTGATCATCT TCATGCTCGGCGCCAACGACATGAAGCCTTGGATCCACGGCAATCCGGTC GCAGCCAAGCAAGGCATCCAGCGGTTGATCGACATCGTGCGTGGTCACGA CTACCCGTTCGACTGGCCGGCGCCGCAGATCCTGATCGTCGCGCCGCCTG TAGTCAGCCGCACCGAAAATGCCGACTTCAAGGAAATGTTCGCCGGTGGC GATGACGCCTCGAAGTTTTTGGCACCGCAATATGCCGCGCTCGCCGACGA AGCCGGCTGTGGCTTCTTCGACGCCGGCAGCGTGGCCCAAACCACACCGC TCGATGGCGTTCACCTCGATGCCGAAAACACGCGAGAAATCGGCAAGGCG CTGACGCCGATCGTGCGCGTCATGCTGGAATTGTAA Mesorhizobium loti Mlo I (Q98MY5) (Amino Acid) (SEQ ID NO: 97) MKTVLCYGDSLTWGYNAEGGRHALEDRWPSVLQAALGAGVQVIADGLNGR TTAFDDHLAGADRNGARLLPTVLTTHAPIDLIIFMLGANDMKPWIHGNPV AAKQGIQRLIDIVRGHDYPFDWPAPQILIVAPPVVSRTENADFKEMFAGG DDASKFLAPQYAALADEAGCGFFDAGSVAQTTPLDGVHLDAENTREIGKA LTPIVRVMLEL Moraxella bovis Mbo (AAK53448) (DNA) (SEQ ID NO: 98) ATGAAAAAATCCGCCTTTGCCAAATACTCAGCACTTGCCCTAATGGTTGG GATGTGCCTGCACACCGCTTACGCCAAGGAGTTTAGCCAAGTCATCATTT TTGGGGACAGCTTGTCCGATACAGGTCGCCTAAAAGATATGGTCGCCCGA AAAGATGGCACCCTTGGCAACACCTTACAGCCATCTTTTACCACCAACCC CGACCCTGTATGGTCAAGCTTATTTGCCCAAAGTTATGGCAAAACCGCCA GTCCCAACACGCCTGACAATCCCACTGGCACTAACTATGCCGTGGGCGGA GCTCGCTCTGGCTCGGAGGTCAATTGGAATGGTTTTGTGAATGTACCCTC CACCAAAACGCAAATCACCGACCATTTGACCGCCACAGGTGGCAAAGCCG ACCCTAATACCCTGTATGCCATTTGGATTGGCTCTAATGACTTAATTTCA GCTTCTCAAGCCACCACAACAGCCGAAGCCCAAAACGCCATTAAAGGTGC GGTAACTCGCACCGTGATAGACATCGAAACACTCAATCAAGCAGGGGCGA CAACCATTTTGGTGCCAAATGTGCCTGATTTGAGCCTCACGCCCCGAGCC ATCTATGGCGAAAGCCTCATGGCAGGCGTGCAAGACAAAGCCAAACTCGC CTCAAGTCTGTATAATAGCGGTCTGTTTGAAGCATTAAATCAATCCACCG CCAACATCATCCCTGCCAACACCTTTGCCCTACTCCAAGAAGCGACCACA AATAAAGAAGCCTTTGGTTTTAAAAACACGCAAGGCGTGGCGTGTCAAAT GCCCGCTCGTACCACAGGGGCGGATGATGTGGCTTCTACTTCCTTGGCAT GTACCAAAGCCAATCTTATAGAAAACGGGGCAAATGACACCTACGCCTTT GCCGATGACATTCACCCATCGGGACGCACGCACCGCATTTTGGCACAGTA TTACCGTTCTATCATGGACGCCCCTACTCACATGGGTAAACTCTCAGGCG AGCTTGTCAAAACAGGTTCAGCCCACGACCGTCATGTTTACCGTCAGCTT GACAGGCTTAGTGGCTCACAGCACAGCATTTGGGCAAACGTCTATGCCAG CGACCGTACCGACCCCACCACCCAAATCGGCTTGGACGTGGCAGGTTCAT CAAGCCATACAGGGGCGTATCTGAGCCACCAAAACCAAGATTATGTGCTG GATGACACCCTATCATCAGATGTCAAAACCATTGGCATGGGGCTGTATCA TCGCCATGACATCGGCAATGTCCGTCTAAAAGGCGTGGCAGGTATCGACC GACTTAGCGTGGATACGCACCGCCATATCGACTGGGAGGGGACAAGCCGT TCGCACACCGCAGATACCACCGCCAGACGTTTTCATGCAGGGCTACAAGC CAGCTATGGCATAGACATGGGCAAAGCCACCGTGCGTCCGCTTATCGGCG TACATGCCCAAAAAGTCAAAGTAAATGACATGACCGAGAGCGAATCAACT TTATCCACCGCCATGCGTTTTGGCGAGCAAGAACAAAAGTCCCTACAAGG CGAGATTGGCGTCGATGTGGCTTATCCGATTAGCCCTGCTTTGACTCTGA CGGGCGGTATCGCTCACGCTCATGAGTTTAACGATGATGAACGCACCATT AATGCCACTTTAACCTCCATTCGTGAATACACGAAGGGCTTTAATACAAG CGTTAGCACCGACAAATCTCACGCCACCACCGCTCATCTGGGCGTACAAG GGCAACTTGGCAAGGCAAATATTCATGCAGGCGTTCACGCCACCCACCAA GACAGCGATACAGACGTGGGTGGTTCGCTTGGGGTTCGCTTGATGTTTTA A Moraxella bovis Mbo (AAK53448) (Amino Acid) (SEQ ID NO: 99) MKKSAFAKYSALALMVGMCLHTAYAKEFSQVIIFGDSLSDTGRLKDMVAR KDGTLGNTLQPSFTTNPDPVWSSLFAQSYGKTASPNTPDNPTGTNYAVGG ARSGSEVNWNGFVNVPSTKTQITDHLTATGGKADPNTLYAIWIGSNDLIS ASQATTTAEAQNAIKGAVTRTVIDIETLNQAGATTILVPNVPDLSLTPRA IYGESLMAGVQDKAKLASSLYNSGLFEALNQSTANIIPANTFALLQEATT NKEAFGFKNTQGVACQMPARTTGADDVASTSLACTKANLIENGANDTYAF ADDIHPSGRTHRILAQYYRSIMDAPTHMGKLSGELVKTGSAHDRHVYRQL DRLSGSQHSIWANVYASDRTDPTTQIGLDVAGSSSHTGAYLSHQNQDYVL DDTLSSDVKTIGMGLYHRHDIGNVRLKGVAGIDRLSVDTHRHIDWEGTSR SHTADTTARRFHAGLQASYGIDMGKATVRPLIGVHAQKVKVNDMTESEST LSTAMRFGEQEQKSLQGEIGVDVAYPISPALTLTGGIAHAHEFNDDERTI NATLTSIREYTKGFNTSVSTDKSHATTAHLGVQGQLGKANIHAGVHATHQ DSDTDVGGSLGVRLMF Chromobacterium violaceum Cvi (Q7NRP5) (DNA) (SEQ ID NO: 100) ATGCGCTCTATCGTCTGCAAAATGCTGTTCCCTTTGTTGCTGCTGTGGCA GCTGCCCGCCCTGGCCGCCACCGTGCTGGTGTTCGGCGACAGCCTGTCCG CCGGCTACGGCCTGGCCCCGGGCCAGGGATGGGCGGCGCTGCTGGCGCGC GACCTCTCGCCCCGGCACAAGGTGGTCAACGCCAGCGTGTCCGGCGAAAC CAGCGCCGGCGGCCTGTCCAGGCTGCCCGACGCGCTCGCCCGCCACCAGC CCGACGTGCTGGTGCTGGAACTCGGCGCCAACGATGGCCTGCGCGGCCTG CCGATGGCTGACATGAGGCGCAACCTGCAGCGGATGATAGACCTGGCCCA GGCGCGCAAGGCCAAGGTGCTGCTGGTGGGCATGGCGCTGCCACCCAACT ATGGCCCCCGCTACGGCGCCGAGTTCCGCGCCGTTTATGACGATTTGGCC CGCCGCAACCGCCTGGCCTACGTGCCGCTGCTGGTCGAGGGCTTCGCCGG CGACCTCGGCGCCTTCCAGCCCGACGGCCTGCATCCCCGCGCGGAGAAGC AGGCCACCATGATGCGCACGGTCAAGGCAAAACTGCCAGTGAAATAA Chromobacterium violaceum Cvi (Q7NRP5) (Amino Acid) (SEQ ID NO: 101) MRSIVCKMLFPLLLLWQLPALAATVLVFGDSLSAGYGLAPGQGWAALLAR DLSPRHKVVNASVSGETSAGGLSRLPDALARHQPDVLVLELGANDGLRGL PMADMRRNLQRMIDLAQARKAKVLLVGMALPPNYGPRYGAEFRAVYDDLA RRNRLAYVPLLVEGFAGDLGAFQPDGLHPRAEKQATMMRTVKAKLPVK Vibrio vulnificus Vvu (AA007232) (DNA) (SEQ ID NO: 102) ATGTTTTTCCTTTCTAGCGTCGCACACGCAACCGAGAAAGTGTTAATTCT TGGCGACAGCCTAAGTGCAGGATACAACATGTCTGCAGAGCAGGCTTGGC CTAATTTGTTACCAGAAGCATTGAATACATACGGAAAAAACGTAGAAGTG ATCAACGCCAGTATCTCTGGAGACACAACCGGCAATGGACTATCTCGTCT GCCTGAGTTGTTAAAAACGCACTCACCAGACTGGGTGCTTATTGAGTTGG GTGCCAATGATGGCTTGCGAGGTTTCCCGCATAAAGTGATCTCTTCAAAC CTTTCGCGAATGATTCAACTCAGTAAAGCCTCAGACGCTAAAGTCGCATT GATGCAAATTCGTGTACCGCCTAACTATGGCAAGCGCTACACCGATGCAT TTGTCGAACTCTACCCTACGCTTGCTGAACATCACCAAGTCCCGTTGCTC CCCTTTTTCTTAGAGGAAGTGATCGTGAAACCGGAATGGATGATGCCTGA TGGCTTACACCCAATGCCCGAAGCTCAGCCTTGGATCGCTCAATTTGTTG CAAAAACGTTTTACAAACATCTCTAA Vibrio vulnificus Vvu (AA007232)  (Amino Acid) (SEQ ID NO: 103) MFFLSSVAHATEKVLILGDSLSAGYNMSAEQAWPNLLPEALNTYGKNVEV INASISGDTTGNGLSRLPELLKTHSPDWVLIELGANDGLRGFPHKVISSN LSRMIQLSKASDAKVALMQIRVPPNYGKRYTDAFVELYPTLAEHHQVPLL PFFLEEVIVKPEWMMPDGLHPMPEAQPWIAQFVAKTFYKHL Ralstonia eutropha Reu (ZP00166901) (DNA) (SEQ ID NO: 104) ATGCCATTGACCGCGCCGTCTGAAGTCGATCCGCTGCAAATCCTGGTCTA TGCCGATTCGCTTTCGTGGGGCATCGTGCCCGGCACCCGCCGGCGGCTTC CCTTCCCGGTTCGCTGGCCAGGCCGGCTCGAACTCGGCCTGAACGCCGAC GGCGGCGCCCCGGTCCGCATCATCGAGGACTGCCTGAACGGCCGGCGCAC CGTCTGGGACGACCCATTCAAACCGGGCCGCAACGGCTTGCAAGGGCTGG CGCAGCGCATCGAGATCCATTCCCCGGTGGCGCTCGTGGTTTTGATGCTG GGCAACAACGATTTCCAGTCCATGCATCCGCACAACGCCTGGCATGCGGC ACAGGGCGTCGGCGCGCTGGTCCACGCCATCCGGACGGCGCCGATCGAAC CGGGAATGCCGGTGCCGCCGATCCTGGTGGTGGTGCCGCCGCCGATCCGC ACGCCCTGCGGGCCGCTCGCGCCCAAGTTCGCCGGCGGCGAACACAAGTG GGCAGGCCTGCCCGAGGCGCTGCGCGAACTGTGCGCCACTGTCGACTGCT CGCTGTTCGATGCGGGTACCGTGATCCAGAGCAGTGCCGTCGACGGCGTA CACCTTGACGCCGATGCCCATGTCGCCCTGGGCGATGCCCTGCAACCGGT CGTTCGTGCGCTGCTCGCCGAATCCTCGGGACATCCCTCCTAA Ralstonia eutropha Reu (ZP00166901)  (Amino Acid) (SEQ ID NO: 105) MPLTAPSEVDPLQILVYADSLSWGIVPGTRRRLPFPVRWPGRLELGLNAD GGAPVRIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALVVLML GNNDFQSMHPHNAWHAAQGVGALVHAIRTAPIEPGMPVPPILVVVPPPIR TPCGPLAPKFAGGEHKWAGLPEALRELCATVDCSLFDAGTVIQSSAVDGV HLDADAHVALGDALQPVVRALLAESSGHPS Salmonella typhimurium Stm (AAC38796) (DNA) (SEQ ID NO: 106) ATGACCCAAAAGCGTACCCTGCTAAAATACGGCATACTCTCGCTGGCGCT GGCCGCGCCATTATCTGCCTGTGCGTTTGACTCTCTTACGGTGATTGGCG ATAGCCTTAGCGATACCGGTAATAACGGTCGCTGGACCTGGGATAGTGGT CAAAATAAGCTCTACGACGAACAGTTGGCCGAACGATATGGGCTGGAATT AAGCCCTTCCAGCAATGGCGGCTCTAATTATGCCGCCGGCGGCGCGACGG CGACCCCGGAATTAAACCCGCAGGATAATACCGCGGATCAGGTACGGCAG TGGCTTGCCAAAACGGGGGGAAAAGCCGACCACAACGGTTTGTATATTCA CTGGGTCGGCGGAAACGATCTGGCGGCGGCCATCGCGCAACCAACCATGG CACAGCAAATAGCCGGTAATAGCGCCACTAGCGCGGCGGCGCAGGTAGGG CTGTTACTGGATGCCGGCGCCGGGCTGGTCGTGGTGCCAAACGTACCGGA TATTAGTGCGACGCCAATGCTTCTGGAGGCGGTAATCACCGCTGGGCTGG GCGCAGCGGCGCCCCCGGCGCTAAAAGCGGCGTTAGATGCGCTGGCGGAG GGCGCTACGCCCGATTTCGCCAGTCGGCAACAGGCGATCCGCAAGGCGCT GCTGGCGGCGGCTGCAACGGTAAGCAGCAATCCATTTATTCAGCAACTGC TCGTTGAACAACTGCTGGCGGGCTATGAAGCGGCGGCAGGGCAGGCGTCA GCTCTGACCGATTATTATAATCAGATGGAAGAGAAGGGGCTGGAGCAACA CGGCGGCAATATAGCCCGTGCCGATATCAACGGCCTCTTTAAGGAAATTC TTGCCAACCCGCAGGCGTTTGGTCTGACAAATACCGTAGGTATGGCCTGC CCGCCTGGCGTATCCGCTTCGGCGTGCTCCTCGGCAATGCCTGGATTTAA TGCGTCGCAGGACTATGTGTTTGCCGATCATTTACATCCCGGTCCGCAGG TCCATACCATTATTGCGCAATATATTCAGTCGATCATTGCCGCGCCGGTA CAGGCGACATACCTGAACCAAAGCGTTCAGTCGATGGCGCAAGGCAGTCG TACCACGCTTGACAGCCGTTATCAGCAGCTTCGCCAGGGGGAAAATCCTG TTGGTTCGCTGGGCATGTTCGGCGGATACAGCGGGGGATATCAACGTTAT GATAATAATGAGGCCGACGGGAACGGTAATCATAATAATCTGACGGTTGG CGTCGATTATCAGCTTAACGAGCAGGTTCTGCTGGGAGGGCTGATAGCCG GTTCTCTGGATAAGCAACATCCTGACGATAATTATCGTTATGATGCCCGC GGTTTTCAGGCCGCCGTATTCAGCCATTTACGCGCCGGTCAGGCGTGGCT GGATAGCGATTTACACTTTCTGTCCGCTAAATTCAGTAACATTCAGCGCA GTATAACGCTCGGTGCGCTAAGACGGGTGGAAGAGGGCGAAACCAACGGT CGGCTGTCGGGCGCGAGCTTAACCAGCGGTTATGATTTTGTCATGGTGCC GTGGTTAACGACCGGACCGATGCTGCAATATGCATGGGATTACAGCCACG TTAATGGTTATAGCGAGAAGCTCAATACCAGTACATCAATGCGTTTTGGT GACCAAAACGCCCATTCGCAGGTGGGTAGCGCGGGTTGGCGTCTGGATCT TCGCCACAGCATCATTCACTCCTGGGCGCAGATTAATTATCGCCGTCAGT TTGGCGATGATACGTATGTGGCGAACGGCGGCCTTAAATCGACCGCGCTG ACGTTTAGCCGCGACGGAAAAACGCAGGATAAAAACTGGGTTGATATCGC GATTGGCGCAGATTTTCCGCTGTCGGCAACGGTGTCCGCTTTCGCCGGGC TGTCGCAAACGGCAGGGTTAAGCGATGGCAATCAAACCCGTTATAACGTT GGGTTTAGCGCCCGATTTTAA Salmonella typhimurium Stm (AAC38796) (Amino Acid) (SEQ ID NO: 107) MTQKRTLLKYGILSLALAAPLSACAFDSLTVIGDSLSDTGNNGRWTWDSG QNKLYDEQLAERYGLELSPSSNGGSNYAAGGATATPELNPQDNTADQVRQ WLAKTGGKADHNGLYIHWVGGNDLAAAIAQPTMAQQIAGNSATSAAAQVG LLLDAGAGLVVVPNVPDISATPMLLEAVITAGLGAAAPPALKAALDALAE GATPDFASRQQAIRKALLAAAATVSSNPFIQQLLVEQLLAGYEAAAGQAS ALTDYYNQMEEKGLEQHGGNIARADINGLFKEILANPQAFGLTNTVGMAC PPGVSASACSSAMPGFNASQDYVFADHLHPGPQVHTIIAQYIQSIIAAPV QATYLNQSVQSMAQGSRTTLDSRYQQLRQGENPVGSLGMFGGYSGGYQRY DNNEADGNGNHNNLTVGVDYQLNEQVLLGGLIAGSLDKQHPDDNYRYDAR GFQAAVFSHLRAGQAWLDSDLHFLSAKFSNIQRSITLGALRRVEEGETNG RLSGASLTSGYDFVMVPWLTTGPMLQYAWDYSHVNGYSEKLNTSTSMRFG DQNAHSQVGSAGWRLDLRHSIIHSWAQINYRRQFGDDTYVANGGLKSTAL TFSRDGKTQDKNWVDIAIGADFPLSATVSAFAGLSQTAGLSDGNQTRYNV GFSARF

In total, nine of the new “GDSL”-type esterases were identified in 6 metagenomic libraries and BRAIN's esteraselipase library. Eight of these genes were heterologously expressed in E. coli and the resulting enzymes analyzed for activity in the assays described herein. The characterization of these enzymes for perhydrolase activity revealed that one displayed the desired actibity. A second one was predicted to show this actibity due t the presence of amino acids conserved amoung this group of enzymes.

Comparison of the sequences of enzymes for which the presence or absence of the desired perhydrolase activity was determined led to the identification of 19 amino acid positions which were conserved among the enzymes which displayed the desired perhydrolase activity. Thus, it is contemplated that these conserved amino acids are essential for the perhydrolase reaction and/or is a structural feature of perhydrolase enzymes.

One of the structural motifs (“G/ARTT”) conserved among esterases with the desired perhydrolase activity was used to design degenerate primers which provided the means to focus the screening on true perhydrolases among “GDSL”-type esterases. Indeed, the use of these “G/ARTT” primers led to the identification of enzymes with the desired perhydrolase activity from the metagenome. However, it is not intended that the use of the metagenome be limited to any particular assay method. Indeed, it is contemplated that the metagenome be searched by assaying for a particular enzyme activity or activities desired (e.g., perhydrolysis and/or acyltransferase (cofactor dependent or independent) activity). In addition, screening using poly and/or monoclonal anti-sera directed against a protein of interest finds use in the present invention. In additional embodiments, the metagenome is searched using degenerate primer sets based on the sequence of the protein of interest.

In addition, the knowledge of the structure/function relationship of perhydrolases allowed searching for these enzymes in genome sequences of cultivable microorganisms. “GDSL”-type esterases identified in different bacterial isolates, the corresponding genes of 10 enzymes were amplified and heterologously expressed in E. coli. The resulting enzyme samples of seven clones were analyzed using the assays described herein. Of five samples characterized to date, 4 enzymes indeed showed the desired activity and all results confirmed the proposed relationship between primary structural determinants and the function of perhydrolases. Thus, an enzyme library of 19, “GDSL”-type esterases comprising at least 6 perhydrolases with the desired perhydrolase activity was set up. The identified correlation between the structure and function of perhydrolases provides a definition of the sequence space used by enzymes with the desired perhydrolase activity.

Comparisons were made of protein sequences of enzymes for which the absence or presence of the desired perhydrolase activity. This revealed a correlation between the presence of certain amino acids and the capability to perform perhydrolase reactions. This knowledge was used to identify enzymes containing these conserved amino acids in sequenced genomes from cultivable microorganisms. The following enzymes were identified and experiments to amplify the genes from the genomic DNA of the corresponding strains using specific primers were performed.

TABLE 1 “GDSL”-type Esterases with a “GRTT”-Motif From Bacterial Isolates Protein Ac- Expression Isolate Identifier ronym Amplicon Vector Sinorhizobium Sma1993 Sme I yes pLO_SmeI meliloti Sinorhizobium Q92XZ1 Sme II yes pET26_SmeII meliloti Sinorhizobium Q9EV56 Sme III yes pET26_SmeIII meliloti Agrobacterium Q9KWB1 Arh I no — rhizogenes Agrobacterium Q9KWA6 Arh II no — rhizogenes Agrobacterium AAD02335 Atu III yes pET26_AtuIII tumefaciens Mesorhizobium Q98MY5 Mlo I yes pET26_Mlo loti Mesorhizobium ZP_00197751 Mlo II no — loti Ralstonia Q8XQI0 Rso no — solanacearum Ralstonia ZP_00166901 Reu yes n.d. eutropha Moraxella bovis AAK53448 Mbo yes pET26_Mbo Burkholderia ZP_00216984 Bce no — cepacia Chromobacterium Q7NRP5 Cvi yes pET26_Cvi violaceum Pirellula sp. NP_865746 Psp n.d. n.d. Vibrio vulnificus AA007232 Vvu yes pET26_Vvu Salmonella AAC38796 Sty yes pET26_Sty typhimurium

In the cases of A. rhizogenes, M. loti (enzyme II), R. solanacearum and B. cepacia no amplicon could be generated. It was thought that this was probably due to genetic differences between the strains used in this investigation and those used for the sequencing of the genes deposited in the public domain databases. One reason might be that the corresponding genes are located on plasmids which are not present in the strains used in this investigation. However, it is not intended that the present invention be limited to any particular mechanism or theory.

The amplicons from all other strains were sequenced. In many cases there were differences between the sequence from the databases and the sequence determined during the development of the present invention. By sequencing two clones from independent amplifications, mutations introduced by the polymerase could be nearly excluded. The sequences of the genes and the deduced amino acid sequences of “GDSL”-type esterases with a “GRTT”-motif or variations from bacterial isolates are provided below:

SMa1993_(—) Sinorhizobium meliloti (Sme I) (SEQ ID NOS:88 and 89) Q92XZ1_(—) Sinorhizobium meliloti (Sme II) (SEQ ID NOS:90 and 91) Q9EV56_(—) Sinorhizobium meliloti (Sme III) (SEQ ID NOS:92 and 93) AAD02335_(—) Agrobacterium tumefaciens (Atu III) (SEQ ID NOS: 94 and 95) Q98MY5_(—) Mesorhizobium loti (Mlo I) (SEQ ID NOS:96 and 97) ZP_(—)00166901_(—) Ralstonia eutropha (Reu) (SEQ ID NOS:104 and 105) AAK53448_(—) Moraxella bovis (Mbo) (SEQ ID NOS: 98 and 99) Q7NRP5_(—) Chromobacterium violaceum (Cvi) (SEQ ID NOS:100 and 101) AA007232_(—) Vibrio vulnificus (Vvu) (SEQ ID NOS:102 and 103) AAC38796_(—) Salmonella typhimurium (Stm) (SEQ ID NOS:106 and 107)

Q9KWB1_Agrobacterium rhizogenes (Arh I) (SEQ ID NO: 108) MICHKGGEEMRSVLCYGDSNTHGQIPGGSPLDRYGPNERWPGVLRRELGS QWYVIEEGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHAILDLVIIMLGT NDLKARFGQPPSEVAMGIGCLVYDIRELAPGPGGKPPEIMVVAPPPMLDD IKEWEPIFSGAQEKSRRLALEFEIIADSLEVHFFDAATVASCDPCDGFHI NREAHEALGTALAREVEAIGWR (SEQ ID NO: 109) ATGATTTGCCATAAAGGTGGGGAGGAAATGCGGTCAGTCTTATGCTACGG CGACTCGAATACGCACGGCCAGATTCCGGGGGGCTCACCGCTCGACCGAT ACGGGCCGAACGAGCGCTGGCCTGGCGTTTTGAGACGGGAGCTTGGAAGC CAGTGGTATGTGATCGAGGAGGGCCTGAGTGGCCGCACGACGGTTCGCGA CGATCCGATCGAGGGCACGATGAAAAACGGCCGGACCTACCTGCGTCCGT GCCTCATGAGCCACGCGATCCTCGATCTCGTGATTATCATGCTCGGGACG AACGACCTGAAAGCGCGCTTCGGTCAACCGCCATCGGAAGTGGCGATGGG GATCGGCTGCCTCGTCTACGATATCAGGGAGCTGGCGCCCGGACCGGGCG GCAAGCCCCCCGAAATCATGGTGGTTGCTCCGCCGCCGATGCTGGACGAT ATCAAGGAATGGGAACCCATATTTTCCGGCGCCCAGGAGAAATCCCGGCG TCTCGCGCTTGAGTTTGAAATTATTGCTGATTCGCTTGAAGTACACTTCT TTGACGCCGCGACCGTCGCATCGTGTGATCCTTGCGATGGTTTTCACATC AACCGGGAAGCGCATGAAGCCTTGGGAACAGCGCTTGCCAGGGAAGTGGA GGCGATCGGTTGGAGATGATGA Q9KWA6_Agrobacterium rhizogenes (Arh II) (SEQ ID NO: 110) MAESRSILCFGDSLTWGWIPVPESSPTLRYPFEQRWTGAMAAALGDGYSI IEEGLSARTTSVEDPNDPRLNGSAYLPMALASHLPLDLVIILLGTNDTKS YFRRTPYEIANGMGKLAGQVLTSAGGIGTPYPAPKLLIVSPPPLAPMPDP WFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCDGIHFSA ETNITLGHAIAAKVEAIFSQEAKNAAA (SEQ ID NO: 111) ATGGCAGAGAGCCGCTCAATATTATGTTTTGGGGATTCACTCACATGGGG TTGGATTCCGGTACCGGAGTCGTCGCCGACGCTCAGATATCCCTTTGAGC AGCGCTGGACCGGTGCAATGGCTGCGGCACTCGGTGACGGCTATTCAATC ATCGAGGAAGGCCTTTCCGCCCGCACGACCAGCGTCGAGGATCCGAACGA TCCCAGGCTGAACGGCAGCGCCTACCTGCCGATGGCGCTCGCCAGCCATC TGCCGCTCGATCTCGTCATCATCCTTCTCGGCACCAACGACACCAAGTCC TATTTCCGCCGCACGCCCTATGAGATCGCCAACGGCATGGGCAAGCTTGC CGGACAGGTTCTGACCTCGGCCGGCGGGATCGGCACGCCCTACCCTGCCC CGAAGCTTCTGATCGTTTCGCCGCCGCCGCTCGCTCCCATGCCTGACCCG TGGTTCGAAGGCATGTTCGGTGGCGGTTACGAAAAGTCGCTCGAACTCGC AAAGCAGTACAAGGCGCTCGCCAACTTCCTGAAGGTCGACTTCCTCGACG CCGGCGAGTTTGTAAAGACCGACGGCTGCGATGGAATCCATTTCTCCGCC GAGACGAACATCACGCTCGGCCATGCGATCGCGGCGAAGGTCGAAGCGAT TTTCTCACAAGAGGCGAAGAACGCTGCGGCTTAG ZP_00197751_Mesorhizobium loti (Mlo II) (SEQ ID NO: 112) MKTILCYGDSLTWGYDAVGPSRHAYEDRWPSVLQGRLGSSARVIAEGLCG RTTAFDDWVAGADRNGARILPTLLATHSPLDLVIVMLGTNDMKSFVCGRA IGAKQGMERIVQIIRGQPYSFNYKVPSILLVAPPPLCATENSDFAEIFEG GMAESQKLAPLYAALAQQTGCAFFDAGTVARTTPLDGIHLDAENTRAIGA GLEPVVRQALGL (SEQ ID NO: 113) ATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATGATGC CGTCGGACCCATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGG GCTATGATGCCGTCGGACCCTCACGGCATGCTTATGAGGATCGATGGCCC TCCGTACTGCAAGGCCGCCTCGGTAGCAGTGCGCGGGTGATCGCCGAGGG GCTTTGCGGCCGCACAACTGCGTTTGACGACTGGGTCGCTGGTGCGGACC GGAACGGTGCGCGCATCCTGCCGACGCTTCTTGCGACCCATTCACCGCTT GACCTCGTTATCGTCATGCTCGGGACGAACGACATGAAATCGTTCGTTTG CGGGCGCGCTATCGGCGCCAAGCAGGGGATGGAGCGGATCGTCCAGATCA TCCGCGGGCAGCCTTATTCCTTCAATTATAAGGTACCGTCGATTCTTCTC GTGGCGCCGCCGCCGCTGTGCGCTACCGAAAACAGCGATTTCGCGGAAAT TTTTGAAGGTGGCATGGCTGAATCGCAAAAGCTCGCGCCGCTTTATGCCG CGCTGGCCCAGCAAACCGGATGCGCCTTCTTCGATGCAGGCACTGTGGCC CGCACGACACCGCTCGACGGTATTCACCTCGATGCTGAAAACACGCGCGC CATTGGTGCCGGCCTGGAGCCGGTGGTCCGCCAAGCGCTTGGATTGTGA Q8XQI0_Ralstonia solanacearum (Rso) (SEQ ID NO: 114) MQQILLYSDSLSWGIIPGTRRRLPFAARWAGVMEHALQAQGHAVRIVEDC LNGRTTVLDDPARPGRNGLQGLAQRIEAHAPLALVILMLGTNDFQAIFRH TAQDAAQGVAQLVRAIRQAPIEPGMPVPPVLIVVPPAITAPAGAMADKFA DAQPKCAGLAQAYRATAQTLGCHVFDANSVTPASRVDGIHLDADQHAQLG RAMAQVVGTLLAQ (SEQ ID NO: 115) ATGCAACAGATCCTGCTCTATTCCGACTCGCTCTCCTGGGGCATCATCCC CGGCACCCGCCGGCGCCTGCCGTTCGCCGCCCGCTGGGCCGGGGTCATGG AACACGCGCTGCAGGCGCAAGGGCACGCCGTGCGCATCGTCGAAGACTGC CTCAATGGACGCACCACGGTGCTCGACGATCCCGCGCGGCCGGGGCGCAA CGGACTGCAGGGGCTCGCGCAGCGGATCGAAGCGCACGCCCCGCTTGCCC TGGTCATCCTGATGCTCGGCACCAACGACTTCCAGGCGATCTTCCGGCAC ACCGCCCAGGACGCGGCGCAAGGCGTGGCGCAGCTGGTGCGGGCCATCCG CCAGGCGCCGATCGAACCCGGCATGCCGGTGCCGCCCGTGCTGATCGTGG TGCCGCCGGCCATCACCGCGCCGGCCGGGGCGATGGCCGACAAGTTTGCC GACGCGCAGCCCAAGTGCGCCGGCCTTGCGCAGGCCTATCGGGCAACGGC GCAAACGCTAGGCTGCCACGTCTTCGATGCGAACAGCGTCACGCCGGCCA GCCGCGTGGACGGCATCCACCTCGATGCCGACCAGCATGCGCAGCTGGGC CGGGCGATGGCGCAGGTCGTCGGGACGCTGCTTGCGCAATAA ZP_00216984 Burkholderia cepacia (Bce) (SEQ ID NO: 116) ATGACGATGACGCAGAAAACCGTGCTCTGCTACGGCGATTCGAACACGCA TGGCACACGCCCGATGACGCATGCTGGCGGACTGGGGCGGTTTGCACGCG AAGAACGCTGGACCGGCGTGCTGGCGCAAACGCTCGGTGCGAGCTGGCGG GTCATTGAAGAAGGGTTGCCCGCGCGTACGACCGTGCATGACGATCCGAT CGAAGGCCGGCACAAGAATGGTTTGTCGTATCTGCGCGCGTGCGTCGAAA GCCACTTGCCCGTCGATGTCGTCGTGCTGATGCTCGGGACCAACGATCTG AAGACACGCTTCTCGGTCACGCCCGCCGACATCGCGACATCGGTCGGCGT ATTGCTTGCCAAGATCGCTGCGTGCGGCGCCGGTCCGTCCGGTGCGTCAC CGAAGCTCGTGCTGATGGCGCCTGCGCCGATCGTCGAGGTCGGATTCCTC GGCGAGATCTTTGCGGGCGGCGCAGCGAAGTCGCGGCAGCTCGCGAAGCG GTACGAACAGGTGGCAAGCGATGCCGGTGCGCACTTTCTCGATGCCGGCG CGATCGTCGAGGTGAGCCCGGTGGATGGCGTTCACTTCGCGGCCGATCAG CATCGTGTGCTCGGGCAGCGGGTCGCTGCCCTTCTGCAGCAGATTGCGTA A (SEQ ID NO: 117) MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFAREERWTGVLAQTLGASWR VIEEGLPARTTVHDDPIEGRHKNGLSYLRACVESHLPVDVVVLMLGTNDL KTRFSVTPADIATSVGVLLAKIAACGAGPSGASPKLVLMAPAPIVEVGFL GEIFAGGAAKSRQLAKRYEQVASDAGAHFLDAGAIVEVSPVDGVHFAADQ HRVLGQRVAALLQQIA NP_865746 Pirellula sp (Psp) (SEQ ID NO: 118) MHSILIYGDSLSWGIIPGTRRRFAFHQRWPGVMEIELRQTGIDARVIEDC LNGRRTVLEDPIKPGRNGLDGLQQRIEINSPLSLVVLFLGTNDFQSVHEF HAEQSAQGLALLVDAIRRSPFEPGMPTPKILLVAPPTVHHPKLDMAAKFQ NAETKSTGLADAIRKVSTEHSCEFFDAATVTTTSVVDGVHLDQEQHQALG TALASTIAEILADC (SEQ ID NO: 119) ATGCATTCAATCCTCATCTATGGCGATTCTCTCAGTTGGGGAATCATTCC CGGCACGCGTCGTCGCTTCGCGTTCCATCAGCGTTGGCCGGGCGTCATGG AGATTGAACTGCGACAAACTGGAATCGATGCCCGCGTCATCGAAGACTGC CTCAATGGCCGACGAACCGTCTTGGAAGATCCAATCAAACCCGGACGCAA TGGCCTGGATGGTTTGCAGCAACGGATCGAAATCAATTCACCTCTGTCAC TGGTCGTGCTCTTTCTGGGGACCAACGATTTCCAGTCCGTCCACGAATTC CATGCCGAGCAATCGGCACAAGGACTCGCACTGCTTGTCGACGCCATTCG TCGCTCCCCTTTCGAACCAGGAATGCCGACACCGAAAATCCTGCTTGTCG CACCACCGACGGTTCACCACCCGAAACTTGATATGGCGGCGAAGTTCCAA AACGCGGAAACGAAATCGACGGGACTCGCAGATGCGATTCGCAAGGTCTC AACAGAACACTCCTGCGAATTCTTCGATGCGGCCACGGTCACCACAACAA GTGTCGTCGACGGAGTCCATCTCGATCAAGAACAACATCAAGCACTCGGT ACCGCACTGGCATCGACAATCGCTGAAATACTAGCAGACTGTTGA

As indicated above, the above sequences are the protein sequences and the coding sequences of “GDSL-type” esterases with a “GRTT”-motif or similar motifs from different bacterial isolates. The DNA sequences represent the target-DNA from which specific primers were deduced. All amplicons were ligated as NdeI/XhoI-fragments to pET26 thereby eliminating the pelB-leader sequence of this vector. All of the “GDSL-type” esterases from these isolates were expressed in E. coli Rosetta (DE3) at 28° C. The expression was induced by addition of 100 μM IPTG at an O.D.₅₈₀=1 and the cells were harvested 20 h after induction. Only the cells expressing the enzymes from M. bovis and S. typhimurium were collected 4 h after induction, since previous experiments had shown that the highest activity could be obtained at this point of time. Table 2 summarizes the expression experiments.

TABLE 2 Expression and Characterization of “GDSL”-type Esterases From Bacterial Isolates for Perhydrolase Activity Per- Expres- Solu- hydro- sion bil- Activ- lase GRTT- Strain Enzyme Level² ity³ ity ⁴ Activity Motif S. meliloti Sme I +++ ++ 5770.0 yes ARTT S. meliloti Sme II +++ +++ 85.0 yes GRTT S. meliloti Sme III +++ ++ 746.5 n.d. GRTT A. tumefaciens Atu III n.d⁵. n.d. n.d. n.d. GRTT M. loti Mlo I +++ ++ 1187.3 yes GRTT M. bovis ¹ Mbo + n.d. 25.2 yes ARTT C. violaceum Cvi + + 2422.7 n.d. GETS V vulnificus Vvu n.d. n.d. n.d. n.d. GDTT R. eutropha Reu n.d. n.d. n.d. n.d. GRRT S. typhimurium ¹ Sty + n.d. 17.2 no SRTT ¹outer membrane localized autotransporter protein ²expression level: + moderate overexpression; ++ strong overexpression; +++ very strong overexpression as judged from SDS-PAGE-analysis ³as judged by SDS-PAGE-analysis ⁴ towards p-nitrophenyl butyrate ⁶not determined

With the exception of the enzyme from S. typhimurium, all other enzymes tested showed the desired perhydrolase activity, confirming the correlation between the presence of certain conserved amino acids an the capability to perform perhydrolase reactions. Although the enzyme from S. typhimurium contains the GRTT-motif, it is different from the other enzymes by the location of this motif downstream from block V. In all other enzymes, this motif is located between block I and III, indicating that it might have a different function in the enzyme from S. typhimurium. Thus, the absence of perhydrolase activity in the enzyme from S. typhimurium also supports the identified structure/function-relationship of the perhydrolases provided by the present invention.

Screening of New “GDSL-Type” Esterases in Metagenome Libraries

i) Library 5279

-   -   The full-length sequence of the gene from clone M75bA2 was         completed, as provided below.

1 tgggcggttt cgcggagtcg agcagggaga gatgctcctg ggtcgtacga gttggtacgg    g  r  f   r  g  v   e  q  g   e  m  l  l   g  r  t   s  w  y 61 aggcatcgtt gaagatctca cgcctgcttg aatgcgcgcg gatatggaac ggaccggccg g  g  i  v   e  d  l   t  p  a   —  m  r  a   d  m  e   r  t  g 121 cgctggcgat cggtgtcggc gtggggctgg cgagcctgag cccggtcgcg ctggcgacgc r  a  g  d   r  c  r   r  g  a   g  e  p  e   p  g  r   a  g  d 181 cgccgcgggg caccgtgccg gtgttcaccc gatcggggac agcctgacgg acgagtattt a  a  a  g   h  r  r   g  v  h   p  i   g  d   s  l   t   d  e  y 241 tgagccgttc ttccagtggg ggttctgcgg gaagtcgtgg gccgagattt tggtggagac f  e  p  f   f  q  w   g  f  c   g  k  s  w   a  e  i   l  v  e 301 ggggcgggcg agcatgggcc cgacggcgca gcaggcgggg atcagcgagc cggagggatg t  g  r  a   s  m  g   p  t  a   q  q  a  g   i  s  e   p  e  g 361 gtcggatccg cggaacacgg ggtatcagca caactgggcg cggtactcgt ggagctcctc w  s  d  p   r  n  t   g  y  q   h  n  w  a   r  y  s   w  s  s 421 agacgcgctg accgaggagt cgccgggggc gacgctgagc gtgctgcttg gggcggagta s  d  a  l   t  e  e   s  p  g   a  t  l  s   v  l  l   g  a  e 481 cgcggtggtg ttcattggga ccaacgactt caatccgtcg tggccggcgt atcagagcgt y  a  v  v   f  i   g   t  n  d    f  n  p  s   w  p  a   y  q  s 541 gtatctgagc cagtggagcg acgagcagat cgacacgtac gtgaacgggg tggtgcagaa v  y  l  s   q  w  s   d  e  q   i  d  t  y   v  n  g   v  v  q 601 catcgcgcag atggtggact cgctgaagtc ggtcggggcg aaggtggtgc ttgcgccgcc n  i  a  q   m  v  d   s  l  k   s  v  g  a   k  v  v   l  a  p 661 ggtggatttt cagttcgcgg ggttcctgcg gaactcatgc ccggatccga tgctgcgcga p  v  d  f   q  f  a   g  f  l   r  n  s  c   p  d  p   m  l  r 721 gcaggcgggt attctgacac ggaagtgcca cgaccgggtg cggtcgatgg cgcggcagaa e  q  a  g   i  l  t   r  k  c   h  d  r  v   r  s  m   a  r  q 781 gcacgtggtg ttcgtggaca tgtggcggct gaaccgcgat ttgttcggca acgggttcgc k  h  v  v   f  v  d   m  w  r   l  n  r  d   l  f  g   n  g  f  841 gatcagctac ggccttcgga acacggtgcg cgtgggggac tcggagatcg ggctgcaact a  i  s  y   g  l  r   n  t  v   r  v  g  d   s  e  i   g  l  q 901 ggccgggctg acgggatcgg cggggctggt tccggacggg atccatccgc agcgggtggt l  a  g  l   t  g  s   a  g  l   v  p   d  g   i  h  p    q  r  v 961 gcaggggatc tgggcgaatg cgttcatcgt gggtctgaac gcgcatgggg cgaacatcgc v  q  g  i   w  a  n   a  f  i   v  g  l  n   a  h  g   a  n  i 1021 gcccatcggc gaggcggaga tgtgcgcgat ggggggggtc gtgtacgggg gaacggacac a  p  i  g   e  a  e   m  c  a   m  g  g  v   v  y  g   g  t  d 1081 gctggcgaac ttcctgccgc cggtcgcggg ctacgtggag gacttccgca acgcggggga t  l  a  n   f  l  p   p  v  a   g  y  v  e   d  f  r   n  a  g 1141 cttcgtgtgc acggcggact tcaaccatga ccttggcgtg acgccgacgg acatcttcgc d  f  v  c   t  a  d   f  n  h   d  l  g  v   t  p  t   d  i  f 1201 gttcatcaac gcgtggttca tgaatgatcc ctcggcgcgg atgagcaacc cggagcacac a  f  i  n   a  w  f   m  n  d   p  s  a  r   m  s  n   p  e  h 1261 gcagatcgag gacatcttcg tgtttctgaa tctgtggctg gtggggtgct gaggcagagt t  q  i  e   d  i  f   v  f  l   n  l  w  i   v  g  c   —  g  r 1321 gggaaggggg tcagcccact tcgcgcgtct ggaagaggat gacggcgacg gagaggaaga v  g  r  g   s  a  h   f  a  r   l  e  e  d   d  g  d   g  e  e

In the sequence of S279_M75bA2 provided above (DNA, SEQ ID NO:80; and amino acid sequence, SEQ ID NO:81), the coding sequence running from position 104 through 1312 is shown on a grey background. Conserved structural motifs are shown underlined and in bold.

The derived amino acid sequence showed the highest homology to a hypothetical protein (Y17D7A.2) from Caenorhabditis elegans (BlastP2; swisspir), although with a very high E-value of 2.5 (i.e., indicating a non-reliable hit). The fact that no esterase is among the homologous proteins identified by the BlastP2-analysis indicates that this enzyme is a rather unusual “GDSL-type” esterase. Furthermore, the enzyme is characterized by unusually long peptides between the N-terminus and the “GDSL”-motif and the “DXXH”-motif of block V (containing the active site aspartic acid and histidine) and the C-terminus. The very C-terminal sequence shows similarity to a membrane lipoprotein lipid attachment site. A corresponding signal sequence of lipoproteins was not identified. The gene encoding M75bA5 was amplified but no further efforts were taken for this enzyme since it did not have the conserved amino acids typical of the perhydrolase of the present invention.

ii) Library 5248

The clone carrying the sequence-tag SP7_(—)3j5h which could have been part of a gene encoding a “GDSL”-type esterase was identified (M31bA11), and the sequence was elongated. This facilitated the determination that this sequence did not encode a “GDSL-type” esterase, because block V could not be identified. The generation of this amplicon can be explained by an “unspecific” hybridization of primer 5 h with the first mismatches at nucleotides 10, 14 and 15 from the 3-terminus of the primer. The sequence showed the highest homology to a hypothetical protein (KO3E5.5) from Caenorhabditis elegans with an E-value of 1.6, indicating a non-reliable hit. The sequence-tag from clone S248_M31bA11 is provided below.

  1 cggaattatc atgctgggtt ttaatgacca gcgcgagagg atcaacgaca acctcgatta    r  n  y   h  a  g   f  -  -  p   a  r  e   d  q  r   q  p  r  l     g  i  i   m  l  g   f  n  d   q  r  e  r   i  n  d   n  l  d      e  l   s  c  w  v   l  m  t   s  a  r   g  s  t  t   t  s  i  61 ctgggacgcc taccactccg tcctgggcga gagacagttt tattccggca attccaagat   l  g  r   l  p  l   r  p  g  r   e  t  v   l  f  r   q  f  q  d y  w  d  a   y  h  s   v  l  g    e r  q  f   y  s  g   n  s  k  t  g  t  p   t  t  p   s  w  a    r d  s   f  l  p  a   i  p  r 121 gttcgtcccc atcaccaaga tcgcggtgaa ggcgcgcaag acccggttca ccaatcagat   v  r  p   h  h  q   d  r  g  e   g  a  q   d  p  v   h  q  s  d m  f  v  p   i  t  k   i  a  v   k  a  r  k   t  r  f   t  n  q  c  s  s   p  s  p  r   s  r  -   r  r  a   r  p  g  s   p  i  r

181 ttttcctcag tccggccgca acgtcgatgt caccaccacg gacggcacac tcccccacgc   f  s  s   v  r  p   q  r  r  c   h  h  h   g  r  h   t  p  p  r i  f  p  q   s  g  r   n  v  d   v  t  t  t   d  g  t   l  p  h  f  f  l   s  p  a  a   t  s  m   s  p  p   r  t  a  h   s  p  t

241 caccatgtcc ctggtcgagc actacatccg ggcctgccgc ctgcgcaccc agatcgttcc   h  h  v   p  g  r   a  l  h  p   g  l  p   p  a  h   p  d  r  s a  t  m  s   l  v  e   h  y  i   r  a  c  r   l  r  t   q  i  v  p  p  c   p  w  s  s   t  t  s   g  p  a   a  c  a  p   r  s  f 301 ggccctgatc gttaacggcg attgcgaagg catgtacagc atctatgtcg gctggtcgaa   g  p  d   r  -  r   r  l  r  r   h  v  q   h  l  c   r  l  v  e p  a  l  i   v  n  g   d  c  e   g  m  y  s   i  y  v   g  w  s  r  p  -   s  l  t  a   i  a  k   a  c  t   a  s  m  s   a  g  r 361 aaccaccaag catgttgttt cacgtgaaac aaagccggtc gaaagcgacg gcatggaatt   n  h  q   a  c  c   f  t  -  n   k  a  g   r  k  r   r  h  g  i k  t  t  k   h  v  v   s  r  e   t  k  p  v   e  s  d   g  m  e  k  p  p   s  m  i  f   h  v  k   q  s  r   s  k  a  t   a  w  n 421 tcccgaactg ggcgaagccg acgacatcac cgaagaaacg cttgagtgtg gccttcccga   s  r  t   g  r  s   r  r  h  h   r  r  n   a  -  v   w  p  s  r f  p  e  l   g  e  a   d  d  i   t  e  e  t   l  e  c   g  l  p  f  p  n   w  a  k  p   t  t  s   p  k  k   r  l  s  v   a  f  p 481 catcgaattg atctcggacg ccgatcttct cgtccttcca ccagcgccga caacattcca   h  r  i   d  i  g   r  r  s  s   r  p  s   t  s  a   d  n  i  p  d  i  e  l   i  s  d   a  d  l   l  v  l  p   p  a  p   t  t  f   t  s  n   -  s  r  t   p  i  f   s  s  f   h  q  r  r   q  h  s  541 aggcgcttga gatgggcggg ttcggtcacg atcttgcgcc gtggacaagg gcaaggtccg   r  r  l   r  w  a   g  s  v  t   i  l  r   r  g  q   g  q  g  p q  g  a  -   d  g  r   v  r  s   r  s  c  a   v  d  k   g  k  v  k  a  l   e  m  g  g   f  g  h   d  l  a   p  w  t  r   a  r  s 601 cagatgatcg acgaggcgcg atcaccgaga tgccgcgacg atctgtcgac gctatgtcac   q  m  i   d  e  a   r  s  p  r   c  r  d   d  l  s   t  l  c  h  r  r  -  s   t  r  r   d  h  r   d  a  a  t   i  c  r   r  y  v   a  d  d   r  r  g  a   i  t  e   m  p  r   r  s  v  d   a  m  s  661 cagcgcatgt ccgacggtgg aatgcaagac aggtnggntn gatcgggg (SEQ ID NO: 120)   q  r  m   s  d  g   g  m  q  d   r  ?  ?   ?  s  g (SEQ ID NO: 121)  t  s  a  c   p  t  v   e  c  k   t  g  ?  ?   d  r   (SEQ ID NO: 122)   p  a  h   v  r  r  w   n  a  r   q  ?  ?   ?  i  g  (SEQ ID NO: 123) 

In the above sequence-tag of the clone S248_M31bA11, the primers 3j and 5 h are indicated. Hybridization between primer and template is indicated by arrows, mismatches by open circles. Putative conserved structural motifs are indicated in bold and underlined.

Several further sequence-tags were generated using different primer pairs of the primers 2 and 5 but none turned out to encode a “GDSL”-type esterases. The screening of this library was completed.

iii) Library M091

The elongation of the amplicon SP3_(—)1j5h, which was identified in the insert-DNA of clone M24dG12 proved that the corresponding sequence does not encode a “GDSL”-type esterase. Whereas the sequence encoding a putative block V (DGTHP; SEQ ID NO:124) was found, the corresponding sequence encoding block I was missing. The amplicon was generated due to an “unspecific” hybridization of primer 1j with the first mismatches at positions 5, 10, 11 and 12 from the 3′-terminus of the primer. The sequence-tag of clone M091_M24dG12 s shown below:

   1 gcctgatggc ttcgagttcg tcgaattcac ctcgccccag cccggcgtgc tggaggcggt      a  -  w   l  r  v   r  r  i  h   l  a  p   a  r  r   a  g  g  g     p  d  g   f  e  f   v  e  f   t  s  p  q   p  g  v   l  e  a     l  m   a  s  s  s   s  n  s   p  r  p   s  p  a  c   w  r  r   61 gtttgaaaag ctgggtttca ccctggtcgc caagcaccgg tccaaggatg tggtgctgta   v  -  k   a  g  f   h  p  g  r   q  a  p   v  q  g   c  g  a  v  v  f  e  k   l  g  f   t  l  v   a  k  h  r   s  k  d   v  v  l  c  l  k   s  w  v  s   p  w  s   p  s  t   g  p  r  m   w  c  c  121 ccgccagaac ggcatcaact tcatcctgaa ccgcgagccc cacagccagg ccgcctactt   p  p  e   r  h  q   l  h  p  e   p  r  a   p  q  p   g  r  l  l y  r  q  n   g  i  n   f  i  l   n  r  e  p   h  s  q   a  a  y  t  a  r   t  a  s  t   s  s  -   t  a  s   p  t  a  r   p  p  t  181 tggtgccgag catggcccct ccgcctgtgg cctggccttc cgtgtgaagg atgcgcataa   w  c  r   a  w  p   l  r  l  w   p  g  l   p  c  e   g  c  a  - f  g  a  e   h  g  p   s  a  c   g  l  a  f   r  v  k   d  a  h  l  v  p   s  m  a  p   p  p  v   a  w  p   s  v  -  r   m  r  i  241 ggcttataac cgcgcgctgg aactgggcgc ccagcccatc gagatcccca ccggccccat   g  l  -   p  r  a   g  t  g  r   p  a  h   r  d  p   h  r  p  h k  a  y  n   r  a  l   e  l  g   a  q  p  i   e  i  p   t  g  p  r  l  i   t  a  r  w   n  w  a   p  s  p   s  r  s  p   p  a  p 

 301 ggaactgcgc ctgcccgcca tcaagggcat tggcggcgcc gcctctgtat ttgatcgacc   g  t  a   p  a  r   h  q  g  h   w  r  r   r  l  c   i  -  s  t m  e  l  r   l  p  a   i  k  g   i  g  g  a   a  s  v   f  d  r  w  n  c   a  c  p  p   s  r  a   l  a  a  p   p  l  y   l  i  d

 361 gctttgaaga cggcaagtcc atctacgaca tcgacttcga gttcatcgaa ggcgtggacc   a  l  k   t  a  s   p  s  t  t   s  t  s   s  s  s   k  a  w  t  p  l  -  r   r  q  v   h  l  r   h  r  l  r   v  h  r   r  r  g  r  f  e   d  g  k  s   i  y  d   i  d  f   e  f  i  e   g  v  d  421 gccgccccgc ggggcatggc ctgaacgaga tcgatcacct cacgcacaac gtgtaccggg   a  a  p   r  g  m   a  -  t  r   s  i  t   s  r  t   t  c  t  g  p  p  p  r   g  a  w   p  e  r   d  r  s  p   h  a  q   r  v  p  r  r  p   a  g  h  g   l  n  e   i  d  h   l  t  h  n   v  y  r  481 gccgcatggg cttctgggcc aacttctacg aaaagctgtt caacttccgc gaaatccgct   a  a  w   a  s  g   p  t  s  t   k  s  c   s  t  s   a  k  s  a  g  p  h  g   l  l  g   q  l  l   r  k  a  v   q  l  p   r  n  p  g  r  m   g  f  w  a   n  f  y   e  k  l   f  n  f  r   e  i  r  541 acttcgacat ccagggcgaa tacacgggcc tgacctccaa ggccatgacc gcgcccgacg   t  s  t   s  r  a   n  t  r  a   -  p  p   r  p  -   p  r  p  t  l  l  r  h   p  g  r   i  h  g   p  d  l  q   g  h  d   r  a  r  y  f  d   i  q  g  e   y  t  g   l  t  s   k  a  m  t   a  p  d  601 gcaagattcg catcccgctg aacgaagagt ccaagcaggg cggcggccag atcgaagaat   a  r  f   a  s  r   -  t  k  s   p  s  r   a  a  a   r  s  k  n r  q  d  s   h  p  a   e  r  r   v  q  a  g   r  r  p   d  r  r  g  k  i   r  i  p  l   n  e  e   s  k  q   g  g  g  q   i  e  e  661 ttttgatgca attcaacggc gagggcattc agcacatcgc gctgatctgc gacaacctgc   f  -  c   n  s  t   a  r  a  f   s  t  s   r  -  s   a  t  t  c i  f  d  a   i  q  r   r  g  h   s  a  h  r   a  d  l   r  q  p  f  l  m   q  f  n  g   e  g  i   q  h  i   a  l  i  c   d  n  l  721 tggacgtggt ggacaagctg ggcatggccg gcgtgcagct ggccaccgcg cccaacgagg   w  t  w   w  t  s   w  a  w  p   a  c  s   w  p  p   r  p  t  r a  g  r  g   g  q  a   g  h  g   r  r  a  a   g  h  r   a  q  r  l  d  v   v  d  k  l   g  m  a   g  v  q   l  a  t  a   p  n  e  781 tctattacga aatgctggac acccgcctgc ccggccacgg ccagccggtg cccgagctgc   s  i  t   k  c  w   t  p  a  c   p  a  t   a  s  r   c  p  s  c g  l  l  r   n  a  g   h  p  p   a  r  p  r   p  a  g   a  r  a  v  y  y   e  m  l  d   t  r  l   p  g  h   g  q  p  v   p  e  l

 841 agtcgcgcgg catcttgctg gacggcacca cggccgacgg cacgcacccg cctgctagct   s  r  a   a  s  c   w  t  a  p   r  p  t   a  r  t   r  l  l  a a  v  a  r   h  l  a   g  r  h   h  g  r  r   h  a  p   a  c  -  q  s  r   g  i  l  l   d  g  t   t  a  d   g  t  h  p   p  a  s

 901 tcagatcttc tccacgccca tgctgggccc ggtgttcttc gaattcatcc agcgcgaggg   s  d  l   l  h  a   h  a  g  p   g  v  l   r  i  h   p  a  r  g l  q  i  f   s  t  p   m  l  g   p  v  f  f   e  f i q r e  f  r  s   s  p  r  p   c  w  a   r  c  s   s  n  s  s   s  a  r  961 cgactaccgc gacggctttg gcgaaggcaa cttcaaggcg ctgttcgagt cgctggaacg   r  l  p   r  r  l   w  r  r  q   l  q  g   a  v  r   v  a  g  t g  d  y  r   d  g  f   g  e  g   n  f  k  a   l  f  e   s  l  e  a  t  t   a  t  a  l   a  k  a   t  s  r   r  c  s  s   r  w  n 1021 cgaccagatc cgccgtggtg tgctgaacac ataagacatc agacatccag ggttaaccct   r  p  d   p  p  w   c  a  e  h   i  r  h   q  t  s   r  v  n  p r  d  q  i   r  r  g   v  l  n   t  -  d  i   r  h  p   g  l  t  a  t  r   s  a  v  v   c  -  t   h  k  t   s  d  i  q   g  -  p 1081 gcacaggtgc ctatactgcg cgctccccgg aactcaaaag gatcccgatg tcgctccgta   a  q  v   p  i  l   r  a  p  r   n  s  k   g  s  r   c  r  s  v l  h  r  c   l  y  c   a  l  p   g  t  q  k   d  p  d   v  a  p  c  t  g   a  y  t  a   r  s  p   e  l  k   r  i  p  m   s  l  r 1141 gcaccctgtt cagcaccctt ttggccggcg cagccactgt cgcgctggcg cagaacccgt   a  p  c   s  a  p   f  w  p  a   q  p  l   s  r  w   r  r  t  r -  h  p  v   q  h  p   f  g  r   r  s  h  c   r  a  g   a  e  p  s  t  l   f  s  t  l   l  a  g   a  a  t   v  a  l  a   q  n  p 1201 ctgcccgctc acatcg (SEQ ID NO: 125)   l  p  a   h  i  (SEQ ID NO: 126) v  c  p  l   t  s (SEQ ID NO: 127)  s a  r  s  h     (SEQ ID NO: 128)

Sequence-tag of the clone M091_M24dG12. The primers 1j and 5 h are indicated in the above sequence-tag of the clone M091_M24dG12. Hybridization between primer and template is indicated by arrows, mismatches by open circles. Putative conserved structural motifs are depicted in bold and underlined.

A further sequence-tag (SP1_(—)2b5h) was generated using the primer pair 2b/5h. A BlastX-analysis of the sequence from this tag yielded the highest homology to an arylesterase from Agrobacterium tumefaciens, with 70% identity. The single clone carrying the corresponding gene was identified (M4aE11) and the full length sequence determined to be as shown below:

1 atgaagacca ttctcgccta tggcgacagc ctgacctatg gggccaaccc gatcccgggc   m  k  t   i  l  a   y   g  d  s   l   t  y   g  a  n   p  i  p  g 61 gggccgcggc atgcctatga ggatcgctgg cccacggcgc tggagcaggg gctgggcggc   g  p  r   h  a  y   e  d  r  w   p  t  a   l  e  q   g  l  g  g 121 aaggcgcggg tgattgccga ggggctgggt ggtcgcacca cggtgcatga cgactggttt   k  a  r   v  i  a   e  g  l  g    g  r  t   t   v  h   d  d  w  f 181 gcgaatgcgg acaggaacgg tgcgcgggtg ctgccgacgc tgctcgagag ccattcgccg   a  n  a   d  r  n   g  a  r  v   l  p  t   l  l  e   s  h  s  p 241 ctcgacctga tcgtcatcat gctcggcacc aacgacatca agccgcatca cgggcggacg   l  d  l   i  v  i   m  l   g  t   n  d   i   k  p  h   h  g  r  t 301 gccggcgagg ccgggcgggg catggcgcgg ctggtgcaga tcatccgcgg gcactatgcc   a  g  e   a  g  r   g  m  a  r   l  v  q   i  i  r   g  h  y  a 361 ggccgcatgc aggacgagcc gcagatcatc ctcgtgtcgc cgccgccgat catcctcggc   g  r  m   q  d  e   p  q  i  i   l  v  s   p  p  p   i  i  l  g 421 gactgggcgg acatgatgga ccatttcggc ccgcacgaag cgatcgccac ctcggtggat   d  w  a   d  m  m   d  h  f  g   p  h  e   a  i  a   t  s  v  d 481 ttcgctcgcg agtacaagaa gcgggccgac gagcagaagg tgcatttctt cgacgccggc   f  a  r   e  y  k   k  r  a  d   e  q  k   v  h  f   f  d  a  g 541 acggtggcga cgaccagcaa ggccgatggc atccacctcg acccggccaa tacgcgcgcc   t  v  a   t  t  s   k  a   d  g   i  h  l    d  p  a   n  t  r  a 601 atcggggcag ggctggtgcc gctggtgaag caggtgctcg gcctgtaa (SEQ ID NO: 129)   i  g  a   g  l  v   p  l  v  k   q  v  l   g  l  — (SEQ ID NO: 130)

In the above sequence, the conserved structural motifs are shown in bold and underlined. The BlastP-analysis with the deduced full length amino acid sequence identified the same hit with a identity of 48%. The primary structure of this enzyme showed the “GRTT”-motif proving the usefulness of the primers directed towards block 2 for the identification of “GRTT”-esterases. The gene was amplified to introduce unique restriction enzyme recognition sites and the absence of second site mutations was confirmed by sequencing. The gene was ligated to pET26 and was expressed in E. coli Rosetta (DE3). The vector map is provided in FIG. 5. Expression and control strains were cultivated in LB in the presence of kanamycin (25 μg/ml), chloramphenicol (12.5 μg/ml), and 1% glucose. At an OD₅₈₀ of 1, expression was induced by addition of 100 μM IPTG. Samples were taken at 2, 4, and 20 hours after induction. Cells were separated from the culture supernatant by centrifugation and after resuspending in sample buffer, they wee incubated for 10 minutes at 90° C. An amount of cells representing an OD₅₈₀ of 0.1 was applied to a 4-12% acryl amide gradient gel, which was stained with Coomassie Brilliant Blue R250.

Strong overexpression of the gene was detected already 2 h after induction with 100 μM IPTG, as determined by SDS-PAGE analysis of crude cell extracts from E. coli Rosetta (DE3) pET26_M4aE11. The amount of protein representing M4aE11 (calculated size 23.2 kDa) increased further over time.

Esterase activity of crude cell extracts from strains expressing the “GDSL”-type esterase M4aE11 was determined. An amount of cells corresponding to an O.D.₅₈₀=2 were resuspended in 200 μl of 5 mM Tris/HCl pH 8.0, and lysed by ultrasonication. Then, 20 μl of each sample were used to determine the esterase activity towards p-nitrophenyl butyrate in a total volume of 200 μl. The activity was corrected for the background activity of the control strain. The activity towards p-nitrophenylbutyrate reached about 125 nmol/ml x min 20 h after induction.

In addition, SDS-PAGE analysis of the soluble and insoluble fraction of crude cell extracts from E. coli Rosetta (DE3) pET26_M4aE11 was conducted. Cells from a culture induced with 100 μM IPTG and harvested 4 h and 20 h after induction were lysed by ultrasonication and separated into soluble and insoluble fraction by centrifugation. Sample buffer was added and directly comparable amounts of soluble and insoluble fractions were applied to a 4-12% acryl amide gradient gel, which was stained with Coomassie Brilliant Blue R250. The results of this analysis of the solubility revealed that M4aE11 is partially (estimated 80%) soluble. The screening of the library M091 was completed.

Thus, in total nine different “GDSL”-type esterases were identified in 6 different large insert metagenomic libraries and the esteraseslipases BRAIN's library comprising more than 4.3 Gbp. Eight of these genes were heterologously expressed in E. coli. The resulting enzyme samples of seven clones were characterized for the desired perhydrolase activity. Two of the enzymes displayed this activity. Table 3 summarizes the screening, expression and characterization of the metagenomic “GDSL”-type esterases.

TABLE 3 Expression and Characterization of Metagenomic “GDSL”-Type Esterases Per- GDSL-type Homol- Expression² Solu- Activ- hydrolase Esterase ogy¹ Level bility³ ity⁴ Activity S248_M2bB11 12.9% ++ + 136 − S248_M40cD4 14.8% +++ ++ 50 −/+⁶ S248_M44aA5 12.4% +++ ++ 75 −/+ S261_M2aA12 36.9% ++ ++ 72 +⁷ S279_M70aE8 11.9% +++ + 167 − S279_M75bA2 5.7% n.d⁵. n.d. n.d. n.d.⁵ M091_M4aE11 33.9% +++ ++ 125 n.d. Est105 4.3% +++ − − n.d. Est114 7.8% n.d. n.d. 13 − ¹identity to the prototype enzyme from M. smegmatis calculated with the dialign algorithm (Morgenstern et al., 1996) ²expression level: + moderate overexpression; ++ strong overexpression; +++ very ³strong overexpression as judged from SDS-PAGE-analysis as judged by SDS-PAGE-analysis ⁴towards p-nitrophenyl butyrate; given as nmol/(ml × min) ⁵not determined ⁶perhydrolysis activity 2x background ⁷perhydrolase activity more than 2x background

Engineering of the Perhydrolase

Based on the structure of the perhydrolase, residues which may alter substrate specificity (e.g., Km, kcat, Vmax, chain length, etc.) and or the multimeric nature of the protein were identified. However, it is not intended that the present invention be limited to any particular residues. Nonetheless, site saturation libraries of residues D10, L12, T13, W14, W16, S54, A55, N94, K97, Y99, P146, W149, F150, I194, F196, are constructed, as well as combinatorial libraries of residues: E51A, Y73A, H81D, T127Q and single mutations of the active site residues D192A, H195A and a site saturation library of the conserved D95. Methods for production of such libraries are known to those skilled in the art and include commercially available kits as the Stratagene Quikchange™ Site-directed mutagenesis kit and/or Quikchange™ Multi-Site-directed mutagenesis kit.

Perhydrolase Activity

The use of enzymes obtained from microorganisms is long-standing. Indeed there are numerous biocatalysts known in the art. For example, U.S. Pat. No. 5,240,835 (herein incorporated by reference) provides a description of the transacylase activity of obtained from C. oxydans and its production. In addition, U.S. Pat. No. 3,823,070 (herein incorporated by reference) provides a description of a Corynebacterium that produces certain fatty acids from an n-paraffin. U.S. Pat. No. 4,594,324 (herein incorporated by reference) provides a description of a Methylcoccus capsulatus that oxidizes alkenes. Additional biocatalysts are known in the art (See e.g., U.S. Pat. Nos. 4,008,125 and 4,415,657; both of which are herein incorporated by reference). EP 0 280 232 describes the use of a C. oxydans enzyme in a reaction between a diol and an ester of acetic acid to produce monoacetate. Additional references describe the use of a C. oxydans enzyme to make chiral hydroxycarboxylic acid from a prochiral diol. Additional details regarding the activity of the C. oxydans transacylase as well as the culture of C. oxydans, preparation and purification of the enzyme are provided by U.S. Pat. No. 5,240,835 (incorporated by reference, as indicated above). Thus, the transesterification capabilities of this enzyme, using mostly acetic acid esters were known. However, the determination that this enzyme could carry out perhydrolysis reaction was quite unexpected. It was even more surprising that these enzymes exhibit very high efficiencies in perhydrolysis reactions. For example, in the presence of tributyrin and water, the enzyme acts to produce butyric acid, while in the presence of tributyrin, water and hydrogen peroxide, the enzyme acts to produce mostly peracetic acid and very little butyric acid. This high perhydrolysis to hydrolysis ratio is a unique property exhibited by the perhydrolase class of enzymes of the present invention and is a unique characteristic that is not exhibited by previously described lipases, cutinases, nor esterases.

The perhydrolase of the present invention is active over a wide pH and temperature range and accepts a wide range of substrates for acyl transfer. Acceptors include water (hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classical acyl transfer). For perhydrolysis measurements, enzyme is incubated in a buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide. Typical substrates used to measure perhydrolysis include esters such as ethyl acetate, triacetin, tributyrin, ethoxylated neodol acetate esters, and others. In addition, the wild type enzyme hydrolyzes nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. Peracid and acetic acid can be measured by the assays described herein. Nitrophenylester hydrolysis is also described.

Although the primary example used during the development of the present invention is the M. smegmatis perhydrolase, any perhydrolase obtained from any source which converts the ester into mostly peracids in the presence of hydrogen peroxide finds use in the present invention.

Substrates

In some preferred embodiments of the present invention, esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are utilized with the perhydrolase enzymes of the present invention. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. In additional embodiments, triacetin, tributyrin, neodol esters, and/or ethoxylated neodol esters serve as acyl donors for peracid formation.

Cleaning and Detergent Formulations

The detergent compositions of the present invention are provided in any suitable form, including for example, as a liquid diluent, in granules, in emulsions, in gels, and pastes. When a solid detergent composition is employed, the detergent is preferably formulated as granules. Preferably, the granules are formulated to additionally contain a protecting agent (See e.g., U.S. application Ser. No. 07/642,669 filed Jan. 17, 1991, incorporated herein by reference). Likewise, in some embodiments, the granules are formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium (See e.g., U.S. Pat. No. 5,254,283, incorporated herein by reference in its entirety). In addition, the perhydrolase enzymes of the present invention find use in formulations in which substrate and enzyme are present in the same granule. Thus, in some embodiments, the efficacy of the enzyme is increased by the provision of high local concentrations of enzyme and substrate (See e.g., U.S. Patent Application Publication US2003/0191033, herein incorporated by reference).

Many of the protein variants of the present invention are useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the protein mutants of the invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents (See e.g., U.S. Pat. Nos. 4,404,128 and 4,261,868). A suitable detergent formulation is that described in U.S. Pat. No. 5,204,015 (previously incorporated by reference). Those in the art are familiar with the different formulations which find use as cleaning compositions. As indicated above, in some preferred embodiments, the detergent compositions of the present invention employ a surface active agent (i.e., surfactant) including anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions. Some surfactants suitable for use in the present invention are described in British Patent Application No. 2 094 826 A, incorporated herein by reference. In some embodiments, mixtures surfactants are used in the present invention.

Suitable anionic surfactants for use in the detergent composition of the present invention include linear or branched alkylbenzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates; alkane sulfonates and the like. Suitable counter ions for anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants that find use in the present invention include quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule.

Nonionic surfactants that find use in the present invention generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.

In some preferred embodiments, the surfactant or surfactant mixture included in the detergent compositions of the present invention is provided in an amount from about 1 weight percent to about 95 weight percent of the total detergent composition and preferably from about 5 weight percent to about 45 weight percent of the total detergent composition. In various embodiments, numerous other components are included in the compositions of the present invention. Many of these are described below. It is not intended that the present invention be limited to these specific examples. Indeed, it is contemplated that additional compounds will find use in the present invention. The descriptions below merely illustrate some optional components.

Proteins, particularly the perhydrolase of the present invention can be formulated into known powdered and liquid detergents having pH between 3 and 12.0, at levels of about 0.001 to about 5% (preferably 0.1% to 0.5%) by weight. In some embodiments, these detergent cleaning compositions further include other enzymes such as proteases, amylases, mannanases, peroxidases, oxido reductases, cellulases, lipases, cutinases, pectinases, pectin lyases, xylanases, and/or endoglycosidases, as well as builders and stabilizers.

In addition to typical cleaning compositions, it is readily understood that perhydrolase variants of the present invention find use in any purpose that the native or wild-type enzyme is used. Thus, such variants can be used, for example, in bar and liquid soap applications, dishcare formulations, surface cleaning applications, contact lens cleaning solutions or products, waste treatment, textile applications, pulp-bleaching, disinfectants, skin care, oral care, hair care, etc. Indeed, it is not intended that any variants of the perhydrolase of the present invention be limited to any particular use. For example, the variant perhydrolases of the present invention may comprise, in addition to decreased allergenicity, enhanced performance in a detergent composition (as compared to the wild-type or unmodified perhydrolase).

The addition of proteins to conventional cleaning compositions does not create any special use limitations. In other words, any temperature and pH suitable for the detergent are also suitable for the present compositions, as long as the pH is within the range in which the enzyme(s) is/are active, and the temperature is below the described protein's denaturing temperature. In addition, proteins of the invention find use in cleaning, bleaching, and disinfecting compositions without detergents, again either alone or in combination with a source of hydrogen peroxide, an ester substrate (e.g., either added or inherent in the system utilized, such as with stains that contain esters, pulp that contains esters etc), other enzymes, surfactants, builders, stabilizers, etc. Indeed it is not intended that the present invention be limited to any particular formulation or application.

Substrates

In some preferred embodiments of the present invention, esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are utilized with the perhydrolase enzymes in the detergent formulations of the present invention. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, in some preferred embodiments, detergents comprising at least one perhydrolase, at least one hydrogen peroxide source, and at least one ester acid are provided.

Hydrolases

In addition to the perhydrolase described herein, various hydrolases find use in the present invention, including but not limited to carboxylate ester hydrolase, thioester hydrolase, phosphate monoester hydrolase, and phosphate diester hydrolase which act on ester bonds; a thioether hydrolase which acts on ether bonds; and α-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase, acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptide hydrolase which act on peptide bonds, all these enzymes having high perhydrolysis to hydrolysis ratios (e.g., >1). Preferable among them are carboxylate ester hydrolase, and peptidyl-peptide hydrolase. Suitable hydrolases include: (1) proteases belonging to the peptidyl-peptide hydrolase class (e.g., pepsin, pepsin B, rennin, trypsin, chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein, gastrisin, cathepsin D, bromelin, keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase, carboxypeptidase A and B, and aminopeptidase); (2) carboxylate ester hydrolase including carboxyl esterase, lipase, pectin esterase, and chlorophyllase; and (3) enzymes having high perhydrolysis to hydrolysis ratios. Especially effective among them are lipases, as well as esterases that exhibit high perhydrolysis to hydrolysis ratios, as well as protein engineered esterases, cutinases, and lipases, using the primary, secondary, tertiary, and/or quaternary structural features of the perhydrolases of the present invention.

The hydrolase is incorporated into the detergent composition as much as required according to the purpose. It should preferably be incorporated in an amount of 0.0001 to 5 weight percent, and more preferably 0.02 to 3 weight percent. This enzyme should be used in the form of granules made of crude enzyme alone or in combination with other enzymes and/or components in the detergent composition. Granules of crude enzyme are used in such an amount that the purified enzyme is 0.001 to 50 weight percent in the granules. The granules are used in an amount of 0.002 to 20 and preferably 0.1 to 10 weight percent. In some embodiments, the granules are formulated so as to contain an enzyme protecting agent and a dissolution retardant material (i.e., material that regulates the dissolution of granules during use).

Cationic Surfactants and Long-Chain Fatty Acid Salts

Such cationic surfactants and long-chain fatty acid salts include saturated or fatty acid salts, alkyl or alkenyl ether carboxylic acid salts, a-sulfofatty acid salts or esters, amino acid-type surfactants, phosphate ester surfactants, quaternary ammonium salts including those having 3 to 4 alkyl substituents and up to 1 phenyl substituted alkyl substituents. Suitable cationic surfactants and long-chain fatty acid salts include those disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. The composition may contain from about 1 to about 20 weight percent of such cationic surfactants and long-chain fatty acid salts.

Builders

In some embodiments of the present invention, the composition contains from about 0 to about 50 weight percent of one or more builder components selected from the group consisting of alkali metal salts and alkanolamine salts of the following compounds: phosphates, phosphonates, phosphonocarboxylates, salts of amino acids, aminopolyacetates high molecular electrolytes, non-dissociating polymers, salts of dicarboxylic acids, and aluminosilicate salts. Examples of suitable divalent sequestering agents are disclosed in British Patent Application No. 2094 826 A, the disclosure of which is incorporated herein by reference.

In additional embodiments, compositions of the present invention contain from about 1 to about 50 weight percent, preferably from about 5 to about 30 weight percent, based on the composition of one or more alkali metal salts of the following compounds as the alkalis or inorganic electrolytes: silicates, carbonates and sulfates as well as organic alkalis such as triethanolamine, diethanolamine, monoethanolamine and triisopropanolamine.

Anti-Redeposition Agents

In yet additional embodiments of the present invention, the compositions contain from about 0.1 to about 5 weight percent of one or more of the following compounds as antiredeposition agents: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose. In some preferred embodiments, a combination of carboxymethyl-cellulose and/or polyethylene glycol are utilized with the composition of the present invention as useful dirt removing compositions.

Bleaching Agents

The use of the perhydrolases of the present invention in combination with additional bleaching agent(s) such as sodium percarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxide adduct and/or a photo-sensitive bleaching dye such as zinc or aluminum salt of sulfonated phthalocyanine further improves the detergent effects. In additional embodiments, the perhydrolases of the present invention are used in combination with bleach boosters (e.g., TAED and/or NOBS).

Bluing Agents and Fluorescent Dyes

In some embodiments of the present invention, bluing agents and fluorescent dyes are incorporated in the composition. Examples of suitable bluing agents and fluorescent dyes are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

Caking Inhibitors

In some embodiments of the present invention in which the composition is powdered or solid, caking inhibitors are incorporated in the composition. Examples of suitable caking inhibitors include p-toluenesulfonic acid salts, xylenesulfonic acid salts, acetic acid salts, sulfosuccinic acid salts, talc, finely pulverized silica, clay, calcium silicate (e.g., Micro-Cell by Johns Manville Co.), calcium carbonate and magnesium oxide.

Antioxidants

The antioxidants include, for example, tert-butyl-hydroxytoluene, 4,4′-butylidenebis(6-tert-butyl-3-methylphenol), 2,2′-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol, distyrenated cresol, monostyrenated phenol, distyrenated phenol and 1,1-bis(4-hydroxy-phenyl)cyclohexane.

Solubilizers

In some embodiments, the compositions of the present invention also include solubilizers, including but not limited to lower alcohols (e.g., ethanol, benzenesulfonate salts, and lower alkylbenzenesulfonate salts such as p-toluenesulfonate salts), glycols such as propylene glycol, acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acid amides, benzoate salts and urea.

In some embodiments, the detergent composition of the present invention are used in a broad pH range of from acidic to alkaline pH. In a preferred embodiment, the detergent composition of the present invention is used in mildly acidic, neutral or alkaline detergent wash media having a pH of from above 4 to no more than about 12.

In addition to the ingredients described above, perfumes, buffers, preservatives, dyes and the like also find use with the present invention. These components are provided in concentrations and forms known to those in the art.

In some embodiments, the powdered detergent bases of the present invention are prepared by any known preparation methods including a spray-drying method and a granulation method. The detergent base obtained particularly by the spray-drying method and/or spray-drying granulation method are preferred. The detergent base obtained by the spray-drying method is not restricted with respect to preparation conditions. The detergent base obtained by the spray-drying method is hollow granules which are obtained by spraying an aqueous slurry of heat-resistant ingredients, such as surface active agents and builders, into a hot space. After the spray-drying, perfumes, enzymes, bleaching agents, inorganic alkaline builders may be added. With a highly dense, granular detergent base obtained such as by the spray-drying-granulation method, various ingredients may also be added after the preparation of the base.

When the detergent base is a liquid, it may be either a homogeneous solution or an inhomogeneous dispersion.

The detergent compositions of this invention may be incubated with fabric, for example soiled fabrics, in industrial and household uses at temperatures, reaction times and liquor ratios conventionally employed in these environments. The incubation conditions (i.e., the conditions effective for treating materials with detergent compositions according to the present invention), are readily ascertainable by those of skill in the art. Accordingly, the appropriate conditions effective for treatment with the present detergents correspond to those using similar detergent compositions which include wild-type perhydrolase.

As indicated above, detergents according to the present invention may additionally be formulated as a pre-wash in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements softening, depilling, pilling prevention, surface fiber removal or cleaning. When the detergent composition is a pre-soak (e.g., pre-wash or pre-treatment) composition, either as a liquid, spray, gel or paste composition, the perhydrolase enzyme is generally employed from about 0.00001% to about 5% weight percent based on the total weight of the pre-soak or pre-treatment composition. In such compositions, a surfactant may optionally be employed and when employed, is generally present at a concentration of from about 0.0005 to about 1 weight percent based on the total weight of the pre-soak. The remainder of the composition comprises conventional components used in the pre-soak (e.g., diluent, buffers, other enzymes (proteases), etc.) at their conventional concentrations.

Cleaning Compositions Comprising Perhydrolase

The cleaning compositions of the present invention may be advantageously employed for example, in laundry applications, hard surface cleaning, automatic dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin. However, due to the unique advantages of increased effectiveness in lower temperature solutions and the superior color-safety profile, the enzymes of the present invention are ideally suited for laundry applications such as the bleaching of fabrics. Furthermore, the enzymes of the present invention find use in both granular and liquid compositions.

The enzymes of the present invention also find use in cleaning additive products. Cleaning additive products including the enzymes of the present invention are ideally suited for inclusion in wash processes where additional bleaching effectiveness is desired. Such instances include, but are not limited to low temperature solution cleaning applications. The additive product may be, in its simplest form, one or more of the enzymes of the present invention. Such additive may be packaged in dosage form for addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired. Such single dosage form may comprise a pill, tablet, gelcap or other single dosage unit such as pre-measured powders or liquids. A filler or carrier material may be included to increase the volume of such composition. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Filler or carrier materials for liquid compositions may be water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. The compositions may contain from about 5% to about 90% of such materials. Acidic fillers can be used to reduce pH. Alternatively, the cleaning additive may include activated peroxygen source defined below or the adjunct ingredients as defined below.

The cleaning compositions and cleaning additives of the present invention require an effective amount of the enzymes provided by the present invention. The required level of enzyme may be achieved by the addition of one or more species of the M. smegmatis perhydrolase, variants, homologues, and/or other enzymes or enzyme fragments having the activity of the enzymes of the present invention. Typically, the cleaning compositions of the present invention comprise at least 0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 to about 0.5, or even from about 0.01 to about 0.1 weight percent of at least one enzyme of the present invention.

In some embodiments, the cleaning compositions of the present invention comprise a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, said peroxygen source being selected from the group consisting of:

(i) from about 0.01 to about 50, from about 0.1 to about 20, or even from about 1 to 10 weight percent of a per-salt, an organic peroxyacid, urea hydrogen peroxide and mixtures thereof;

(ii) from about 0.01 to about 50, from about 0.1 to about 20, or even from about 1 to 10 weight percent of a carbohydrate and from about 0.0001 to about 1, from about 0.001 to about 0.5, from about 0.01 to about 0.1 weight percent carbohydrate oxidase; and

(iii) mixtures thereof.

Suitable per-salts include those selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof.

The carbohydrate may be selected from the group consisting of mono-carbohydrates, di-carbohydrates, tri-carbohydrates, oligo-carbohydrates and mixtures thereof. Suitable carbohydrates include carbohydrates selected from the group consisting of D-arabinose, L-arabinose, D-Cellobiose, 2-Deoxy-D-galactose, 2-Deoxy-D-ribose, D-Fructose, L-Fucose, D-Galactose, D-glucose, D-glycero-D-gulo-heptose, D-lactose, D-Lyxose, L-Lyxose, D-Maltose, D-Mannose, Melezitose, L-Melibiose, Palatinose, D-Raffinose, L-Rhamnose, D-Ribose, L-Sorbose, Stachyose, Sucrose, D-Trehalose, D-Xylose, L-Xylose and mixtures thereof.

Suitable carbohydrate oxidases include carbohydrate oxidases selected from the group consisting of aldose oxidase (IUPAC classification EC1.1.3.9), galactose oxidase (IUPAC classification EC1.1.3.9), cellobiose oxidase (IUPAC classification EC1.1.3.25), pyranose oxidase (IUPAC classification EC1.1.3.10), sorbose oxidase (IUPAC classification EC1.1.3.11) and/or hexose oxidase (IUPAC classification EC1.1.3.5), Glucose oxidase (IUPAC classification EC1.1.3.4) and mixtures thereof.

In some preferred embodiments, the cleaning compositions of the present invention also include from about 0.01 to about 99.9, from about 0.01 to about 50, from about 0.1 to 20, or even from about 1 to about 15 weight percent a molecule comprising an ester moiety. Suitable molecules comprising an ester moiety may have the formula:

R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)

wherein R¹ is a moiety selected from the group consisting of H or a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of the present invention, R¹ may comprise from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon atoms, or even from 2 to 100 carbon atoms;

each R² is an alkoxylate moiety, in one aspect of the present invention, each R² is independently an ethoxylate, propoxylate or butoxylate moiety;

R³ is an ester-forming moiety having the formula:

-   -   R⁴CO— wherein R⁴ may be H, substituted or unsubstituted alkyl,         alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and         heteroaryl, in one aspect of the present invention, R⁴ may be         substituted or unsubstituted alkyl, alkenyl, alkynyl, moiety         comprising from 1 to 22 carbon atoms, an aryl, alkylaryl,         alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22         carbon atoms or R⁴ may be a substituted or unsubstituted C₁-C₂₂         alkyl moiety or R⁴ may be a substituted or unsubstituted C₁-C₁₂         alkyl moiety;     -   x is 1 when R¹ is H; when R¹ is not H, x is an integer that is         equal to or less than the number of carbons in R¹     -   p is an integer that is equal to or less than x     -   m is an integer from 0 to 50, an integer from 0 to 18, or an         integer from 0 to 12, and n is at least 1.

In one aspect of the present invention, the molecule comprising an ester moiety is an alkyl ethoxylate or propoxylate having the formula R¹O_(x)[(R²)_(m)(R³)_(n)]_(p) wherein:

-   -   R¹ is an C₂-C₃₂ substituted or unsubstituted alkyl or         heteroalkyl moiety;     -   each R² is independently an ethoxylate or propoxylate moiety;     -   R³ is an ester-forming moiety having the formula:     -   R⁴CO— wherein R⁴ may be H, substituted or unsubstituted alkyl,         alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and         heteroaryl, in one aspect of the present invention, R⁴ may be a         substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety         comprising from 1 to 22 carbon atoms, a substituted or         unsubstituted aryl, alkylaryl, alkylheteroaryl, or heteroaryl         moiety comprising from 4 to 22 carbon atoms or R⁴ may be a         substituted or unsubstituted C₁-C₂₂ alkyl moiety or R⁴ may be a         substituted or unsubstituted C₁-C₁₂ alkyl moiety;     -   x is an integer that is equal to or less than the number of         carbons in R¹     -   p is an integer that is equal to or less than x     -   m is an integer from 1 to 12, and     -   n is at least 1.

In one aspect of the present invention, the molecule comprising the ester moiety has the formula:

R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)

wherein R¹ is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, said R¹ moiety that comprises an amine moiety being selected from the group consisting of a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of Applicants' invention R¹ may comprise from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon atoms, or even from 2 to 100 carbon atoms;

each R² is an alkoxylate moiety, in one aspect of the present invention each R² is independently an ethoxylate, propoxylate or butoxylate moiety;

-   -   R³ is an ester-forming moiety having the formula:         -   R⁴CO— wherein R⁴ may be H, substituted or unsubstituted             alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,             and heteroaryl, in one aspect of the present invention, R⁴             may be a substituted or unsubstituted alkyl, alkenyl, or             alkynyl moiety comprising from 1 to 22 carbon atoms, a             substituted or unsubstituted aryl, alkylaryl,             alkylheteroaryl, or heteroaryl moiety comprising from 4 to             22 carbon atoms or R⁴ may be a substituted or unsubstituted             C₁-C₂₂ alkyl moiety or R⁴ may be a substituted or             unsubstituted C₁-C₁₂ alkyl moiety;     -   x is 1 when R¹ is H; when R¹ is not H, x is an integer that is         equal to or less than the number of carbons in R¹     -   p is an integer that is equal to or less than x     -   m is an integer from 0 to 12 or even 1 to 12, and     -   n is at least 1.

In any of the aforementioned aspects of the present invention, the molecule comprising an ester moiety may have a weight average molecular weight of less than 600,000 Daltons, less than 300,000 Daltons, less than 100,000 Daltons or even less than 60,000 Daltons.

Suitable molecules that comprise an ester moiety include polycarbohydrates that comprise an ester moiety.

The cleaning compositions provided herein will typically be formulated such that, during use in aqueous cleaning operations, the wash water will have a pH of from about 5.0 to about 11.5, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 and about 9.0. Granular laundry products are typically formulated to have a pH from about 9 to about 11. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

When the enzyme(s) of the present invention isare employed in a granular composition or liquid, it may be desirable for the enzyme(s) to be in the form of an encapsulated particle to protect such enzyme from other components of the granular composition during storage. In addition, encapsulation is also a means of controlling the availability of the enzyme(s) during the cleaning process and may enhance performance of the enzyme(s). In this regard, the enzyme(s) may be encapsulated with any encapsulating material known in the art.

The encapsulating material typically encapsulates at least part of the enzyme(s). Typically, the encapsulating material is water-soluble and/or water-dispersible. The encapsulating material may have a glass transition temperature (Tg) of 0° C. or higher. Glass transition temperature is described in more detail in WO 97/1151, especially from page 6, line 25 to page 7, line 2.

The encapsulating material may be selected from the group consisting of carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and combinations thereof. When the encapsulating material is a carbohydrate, it may be typically selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the encapsulating material is a starch. Suitable starches are described in EP 0 922 499; U.S. Pat. No. 4,977,252; U.S. Pat. No. 5,354,559 and U.S. Pat. No. 5,935,826.

The encapsulating material may be a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that can be used are those supplied by Expancel of Stockviksverken, Sweden under the trademark EXPANCEL®, and those supplied by PQ Corp. of Valley Forge, Pa. U.S.A. under the tradename PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL® and SPHERICEL®.

Processes of Making and Using the Cleaning Compositions of the Present Invention

The cleaning compositions of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. No. 5,879,584; U.S. Pat. No. 5,691,297; U.S. Pat. No. 5,574,005; U.S. Pat. No. 5,569,645; U.S. Pat. No. 5,565,422 Del Greco et al.; U.S. Pat. No. 5,516,448; U.S. Pat. No. 5,489,392; and U.S. Pat. No. 5,486,303; all of which are incorporated herein by reference.

Adjunct Materials in Addition to the Enzymes of the Present Invention, Hydrogen Peroxide, and/or Hydrogen Peroxide Source and Material Comprising an Ester Moiety

While not essential for the purposes of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions and may be desirably incorporated in certain embodiments of the invention, for example to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dyes or the like. It is understood that such adjuncts are in addition to the enzymes of the present invention, hydrogen peroxide and/or hydrogen peroxide source and material comprising an ester moiety. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removalanti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348, herein incorporated by reference. The aforementioned adjunct ingredients may constitute the balance of the cleaning compositions of the present invention.

Surfactants—The cleaning compositions according to the present invention may comprise a surfactant or surfactant system wherein the surfactant can be selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof.

The surfactant is typically present at a level of from about 0.1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject cleaning composition.

Builders—The cleaning compositions of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the subject cleaning composition will typically comprise at least about 1%, from about 3% to about 60% or even from about 5% to about 40% builder by weight of the subject cleaning composition.

Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders polycarboxylate compounds. ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.

Chelating Agents—The cleaning compositions herein may contain a chelating agent, Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof.

When a chelating agent is used, the cleaning composition may comprise from about 0.1% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition.

Deposition Aid—The cleaning compositions herein may contain a deposition aid. Suitable deposition aids include, polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as Kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof.

Dye Transfer Inhibiting Agents—The cleaning compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.

When present in a subject cleaning composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the cleaning composition.

Dispersants—The cleaning compositions of the present invention can also contain dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

Enzymes—The cleaning compositions can comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits. Examples of suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, B-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. A typical combination is cocktail of conventional applicable enzymes like protease, lipase, cutinase and/or cellulase in conjunction with amylase.

Enzyme Stabilizers—Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed herein can be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes.

Catalytic Metal Complexes—The cleaning compositions of the present invention may include catalytic metal complexes. One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.

If desired, the compositions herein can be catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in U.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described, for example, in U.S. Pat. No. 5,597,936; and U.S. Pat. No. 5,595,967. Such cobalt catalysts are readily prepared by known procedures, such as taught for example in U.S. Pat. No. 5,597,936, and U.S. Pat. No. 5,595,967.

Compositions herein may also suitably include a transition metal complex of a macropolycyclic rigid ligand—abreviated as “MRL”. As a practical matter, and not by way of limitation, the compositions and cleaning processes herein can be adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and will preferably provide from about 0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor.

Preferred transition-metals in the instant transition-metal bleach catalyst include manganese, iron and chromium. Preferred MRL's herein are a special type of ultra-rigid ligand that is cross-bridged such as 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.

Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00332601, and U.S. Pat. No. 6,225,464.

Method of Use

The cleaning compositions disclosed herein of can be used to clean a situs inter alia a surface or fabric. Typically at least a portion of the situs is contacted with an embodiment of Applicants' cleaning composition, in neat form or diluted in a wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present invention, washing includes but is not limited to, scrubbing, and mechanical agitation. The fabric may comprise most any fabric capable of being laundered in normal consumer use conditions. The disclosed cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5° C. to about 90° C. and, when the situs comprises a fabric, the water to fabric mass ratio is typically from about 1:1 to about 30:1.

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the following abbreviations apply: ° C. (degrees Centigrade); rpm (revolutions per minute); H₂O (water); HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg and ug (micrograms); mg (milligrams); ng (nanograms); μl and ul (microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm and um (micrometer); M (molar); mM (millimolar); μM and uM (micromolar); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl (sodium chloride); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); TAED (N,N,N′N′-tetraacetylethylenediamine); wv (weight to volume); vv (volume to volume); Per (perhydrolase); per (perhydrolase gene); Ms (M. smegmatis); MS (mass spectroscopy); BRAIN (BRAIN Biotechnology Research and Information Network, AG, Zwingenberg, Germany); TIGR (The Institute for Genomic Research, Rockville, Md.); AATCC (American Association of Textile and Coloring Chemists); WFK (wfk Testgewebe GmbH, Bruggen-Bracht, Germany); Amersham (Amersham Life Science, Inc. Arlington Heights, Ill.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); Pierce (Pierce Biotechnology, Rockford, Ill.); Amicon (Amicon, Inc., Beverly, Mass.); ATCC (American Type Culture Collection, Manassas, Va.); Amersham (Amersham Biosciences, Inc., Piscataway, N.J.); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad, Richmond, Calif.); Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Difco (Difco Laboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Novagen (Novagen, Inc., Madison, Wis.); Qiagen (Qiagen, Inc., Valencia, Calif.); Invitrogen (Invitrogen Corp., Carlsbad, Calif.); Genaissance (Genaissance Pharmaceuticals, Inc., New Haven, Conn.); DNA 2.0 (DNA 2.0, Menlo Park, Calif.); MIDI (MIDI Labs, Newark, Del.) InvivoGen (InvivoGen, San Diego, Calif.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co., Biotechnology Systems, Wilmington, Del.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Roche (Hoffmann La Roche, Inc., Nutley, N.J.); Agilent (Agilent Technologies, Palo Alto, Calif.); Minolta (Konica Minolta, Ramsey, N.J.); and Zeiss (Carl Zeiss, Inc., Thornwood, N.Y.).

In the following Examples, various media were used. “TS” medium (per liter) was prepared using Tryptone (16 g) (Difco), Soytone (4 g) (Difco), Casein hydrolysate (20 g) (Sigma), K₂HPO4 (10 g), and d H₂O (to 1 L). The medium was sterilized by autoclaving. Then, sterile glucose was added to 1.5% final concentration. Streptomyces Production Medium (per liter) was prepared using citric acid (H₂O) (2.4 g), Biospringer yeast extract (6 g), (NH₄)2-SO₄ (2.4 g), MgSO₄.7H₂O (2.4 g), Mazu DF204 (5 ml), trace elements (5 ml). The pH was adjusted to 6.9 with NaOH. The medium was then autoclaved to sterilize. After sterilization, CaCl₂.2H₂O (2 mls of 100 mgml solution), KH₂PO4 (200 ml of a 13% (wv) solution at pH6.9), and 20 mls of a 50% glucose solution were added to the medium.

In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions. A reflectometer was used to measure the reflectance of the swatches. Unless otherwise indicated, protein concentrations were estimated by Coomassie Plus (Pierce), using BSA as the standard.

Example 1 Enzyme Analysis

In this Example, methods to assess enzyme purity and activity used in the subsequent Examples and throughout the present Specification are described.

Enzyme Activity Assay (pNB Assay)

This activity was measured by hydrolysis of p-nitrophenylbutyrate. The reaction mixture was prepared by adding 10 ul of 100 mM p-nitrophenylbutyrate in dimethylsulfoxide to 990 ml of 100 mM Tris-HCl buffer, pH 8.0 containing 0.1% triton X-100. The background rate of hydrolysis was measured before the addition of enzyme at 410 nm. The reaction was initiated by the addition of 10 ul of enzyme to 990 ml of the reaction and the change of absorbance at 410 nm was measured at room temperate (˜23° C.). The background corrected results are reported as δA₄₁₀/min/ml or δA₄₁₀/min/mg protein.

Transesterification

Transesterification was measured by GC separation of products in buffered aqueous reactions. Reactions to measure ethyl acetate transesterification with propanol contained in 1 ml of 50 mM KPO4, pH 7.0; 200 mM ethyl acetate, 200 mM 1-propanol, and enzyme. Reactions to measure ethyl acetate transesterification with neopentyl glycol (NPG) contained in 1 ml of 50 mM KPO4, pH 7.0; 303 mM ethyl acetate, 100 mM NPG, and enzyme. The reactions were incubated at the indicated temperatures and for the indicated times. Separations were preformed using a 30M FFAP column (Phenomenex). The inlet split ratio was approximately 1:25, the injector was 250° C., head pressure of 10 psi He, and detection was by FID at 250° C. The chromatography program was 40° C. initial for 4 min, followed by a gradient of 15° C./min to 180° C. Components eluted in the following order and were not quantified; ethyl acetate, ethyl alcohol, propyl acetate, propyl alcohol, acetic acid, NPG diacetate, NPG monoacetate, and NPG.

Perhydrolase Used in Crystallography Studies

This perhydrolase preparation was used for crystallography studies. In addition, unlabelled protein was grown and purified in similar manner. A 500 ml preculture of E. coli BL21(DE3)/pLysS/pMSATNco1-1 was grown in a baffled 2.8 L Fernbach flask on LB containing 100 ug/ml carbenicillin. After overnight culture at 37° C. and 200 rpm on a rotary shaker, the cells were harvested by centrifugation and resuspended in M9 medium containing: glucose, 2 g/L; Na₂HPO₄, 6 g/L; KH₂PO₄, 3 g/L; NH₄Cl, 1 g/L; NaCl, 0.5 g/L; thiamine, 5 mg/L; MgSO₄, 2 mM; CaCl₂, 100 uM; Citric acid.H₂O, 40 mg/L; MnSO₄.H₂O, 30 mg/L; NaCl, 10 mg/L; FeSO₄.7H₂O, 1 mg/L; CoCl₂.6H₂O, 1 mg/L; ZnSO₄.7H₂O, 1 mg/L; CuSO₄.5H₂O, 100 ug/L; H₃BO₃.5H₂O, 100 ug/L; and NaMoO₄.2H₂O, 100 ug/L; and supplemented with carbenicillin, 100 mg/L. The resuspended cells were used to inoculate six Fernbach flasks containing 500 ml each of M9 medium supplemented with carbenicillin (100 mg/L). The cultures were incubated at 20° C. and 200 rpm on a rotary shaker until the OD₆₀₀ reached about 0.7 at which time 100 mg/L of lysine, threonine, and phenylalanine and 50 mg/L of leucine, isoleucine, valine, and selenomethionine were added. After further incubation for 30 min, IPTG was added to a final concentration of 50 uM. The cultures were then incubated overnight (˜15 hr) and harvested by centrifugation. The cell pellet was washed 2 times with 50 mM KPO4 buffer, pH 6.8. The yield was 28.5 μm wet weight of cells to which was added 114 ml of 100 mM KPO4 buffer, pH 8.2 and 5 mg of DNase. This mixture was frozen at −80° C. and thawed 2 times.

The thawed cell suspension was lysed by disruption in a French pressure cell at 20K psi. The unbroken cells and cell membrane material were sedimented by centrifugation at 100K times g for 1 hour. The supernatant crude extract, 128 ml (CE) was then placed in a 600 ml beaker and stirred for 10 minutes in a 55° C. water bath to precipitate unstable proteins. After 10 min the beaker was stirred in ice water for 1 min followed by centrifugation at 15K times g for 15 mM The supernatant from this procedure, HT, contained 118 ml. The HT extract was then made 20% saturating in (NH₄)₂SO₄ by the slow addition of 12.7 g of (NH₄)₂SO₄. This was loaded on to a 10 cm×11.6 cm Fast Flow Phenyl Sepharose (Pharmacia) column equilibrated in 100 mM KPO4 buffer, pH 6.8, containing 20% saturation (109 g/L) (NH₄)₂SO₄. After loading the extract the column was washed with 1700 ml of starting buffer and eluted with a two step gradient. The first step was a linear 1900 ml gradient from start buffer to the same buffer without (NH₄)₂SO₄, the second was a 500 ml elution with 100 mM KPO4, pH 6.8 containing 5% EtOH. Active fractions, 241 ml, were pooled, diluted 100% with water and loaded onto a 1.6 mm×16 mm Poros HQ strong anion exchange column equilibrated in 100 mM Tris-HCl, pH 7.6. After loading the extract, the column was washed with 5 column volumes of starting buffer. The protein was eluted with a 15 column volume gradient from start buffer to start buffer containing 175 mM KCl. The active fractions were pooled and concentrated using a Centriprep 30 (Millipore) to 740 μl. FIG. 6 provides a purification table showing the enzyme activity of the enzyme of the present invention through various steps in the purification process.

The present application must be used to determine the respective values of the parameters of the present invention.

Unless otherwise noted, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.

Enzyme components weights provided herein are based on total active protein. All percentages and ratios were calculated by weight unless otherwise indicated. All percentages and ratios were calculated based on the total composition unless otherwise indicated.

Example 2 Determination of Ratio Between Peracid and Acid Formation

In this Example, methods for determining the ratio of perhydrolysis to hydrolysis are described. In particular, this Example provides methods for determining the ratio between peracid formation (i.e., perhydrolysis) and acid formation (i.e., hydrolysis) resulting from enzyme activity on an ester substrate in the presence of peroxide in an aqueous system.

A. Determination of Perhydrolysis to Hydrolysis Ratio

Preparation of Substrate

The substrates were prepared as described herein. Ethyl acetate (EtOAc) and other water soluble esters were diluted in a desired buffer to a concentration of 10 mM of ester. Tributyrin and other water insoluble substrates were prepared by making substrate swatches. Polyester swatches were cut from non-dyed polyester fabric (Polycotton, PCW 22) using a 58 inch punch and placed in a 24-well microtiter plate (Costar, Cell Culture Plate). The insoluble ester was diluted to 1.03 M in hexane. Then, 10 μL of the insoluble ester solution were then adsorbed onto the polyester swatch.

Determination of Hydrolysis (GC Assay)

The hydrolytic assay described below was used to determine the amount of substrate hydrolysis. In this assay, the assay solution was comprised of 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, and 20 mM potassium chloride in a total volume of 0.99 ml and an amount of enzyme that would generate 20 nmoles of acetic acid per minute at 25° C.

For measuring water insoluble ester hydrolysis, the reaction mixture was added to the insoluble ester fabric swatch. The swatch was prepared as described above (“Preparation of Substrate”). All the other conditions for the assay were the same except for exclusion of other ester substrates.

Hydrolytic activity was measured by monitoring the increase of acids generated by the enzyme from acyl donor substrates using gas chromatography coupled with flame ionization detection. The assay was conducted by first pipetting 50 μL of assay solution containing all the components except the enzyme into 200 mL of methanol (HPLC grade) to determine the amount of acid in the assay solution at time 0. Then, 10 μL of enzyme were added to the assay solution to a desired final concentration which produced approximately 20 nanomoles of acid per minute. A timer was started and 50 μL aliquots were taken from the assay solution and added to 200 μL of methanol at various times, typically 2, 5, 10, 15, 25, 40, and 60 minutes, after addition of the enzyme.

These methanol-quenched samples were then injected into a gas chromatograph coupled with a flame ionization detector (Agilent 6890N) and analyzed for hydrolytic components, acetic, and butyric acids. Gas chromatography was conducted using a nitroterephthalic acid modified polyethylene glycol column (Zebron FFAP; with dimensions: 30 m long, 250 um diameter, 250 nm film thickness). A 3 μL aliquot of sample was applied to the column by a splitless injection under constant a helium flow of 1.0 mL/minute. The inlet was maintained at a temperature of 250° C. and was purged of any remaining sample components after 2 minutes. When analyzing acetic acid, the temperature of the column was maintained at 75° C. for 1 minute after injection, increased 25° C./minute to 100° C., then increased 15° C./minute to 200° C.

When analyzing butyric acid, the temperature of the column was controlled as described above, except the temperature was additionally increased 25° C./minute to 225° C. and held at 225° C. for 1 minute. The flame ionization detector was maintained throughout the chromatography at 250° C. and under constant hydrogen flow of 25 mL/minute, air flow of 200 mL/minute, and a combined column and makeup helium flow of 30 mL/minute. The amount of hydrolyzed acid in the sample was then determined by integrating the acid peak in the chromatogram for total ion counts and calculating the acid from the ion count using a standard curve generated under the above conditions for acetic and butyric acids at varying concentrations in the assay solution (without enzyme).

Determination of Perhydrolysis (OPD Assay)

The perhydrolytic activity assay described below was used to determine the amount of peracid formed in the reaction. In these assays, the solution comprised 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, 20 mM potassium chloride, and 10 mM O-phenylenediamine.

When using water insoluble ester as the acyl donor, an ester adsorbed fabric swatch was used as the substrate, prepared as described above (“Preparation of Substrate”).

Perhydrolytic activity was measured by monitoring the absorbance increase at 458 nm of oxidized O-phenylenediamine (OPD) by peracid generated with the enzyme. The perhydrolytic activity assay solution was prepared in the same manner as the hydrolytic activity assay solution, except that OPD was added to the assay solution to a final concentration of 10 mM. The OPD solution was prepared immediately before conducting the assay by dissolving 72 mg OPD (Sigma-Aldrich, dihydrochloride) in 19.94 mL of the same buffer and the pH was adjusted by slowly adding 60 μL of 13.5 M potassium hydroxide. The pH was measured and if needed, small quantities of potassium hydroxide were added to return the pH to the original pH of the buffer. Then, 495 μL of this OPD solution were added with the other assay components to a final assay volume of 0.990 mL. An assay quenching solution was also prepared by dissolving 36 mg OPD in 20 mL 100 mM citric acid and 70% ethanol.

The assay was typically conducted at 25° C. The assay was started by pipetting 100 μL of assay solution before the addition of the enzyme into 200 μL of quenching solution to determine the amount of perhydrolytic components and background absorbance in the assay solution at time 0. Then, 10 μL of enzyme were added to the assay solution to a desired final concentration which produced approximately 10 nanomoles of peracid per minute. A timer was started and 100 μL aliquots were taken from the assay solution and added to 200 μL of quenching solution at various times, typically 2, 5, 10, 15, 25, 40, and 60 minutes, after adding the enzyme. The quenched assay solutions were incubated for 30 minutes to allow any remaining peracid to oxidize the OPD. Then, 100 μL of each quenched assay solution was transferred to a 96-well microtiter plate (Costar) and the absorbance of the solution was measured at 458 nm by a spectrophotometric plate reader (Molecular Devices, SpectraMAX 250). The amount of peracid in each quenched sample was calculated using a standard curve generated under the above conditions with peracetic acid at varying concentrations in the assay solution (without enzyme).

Perhydrolysis Hydrolysis ratio:

Perhydrolysis Hydrolysis ratio=Perhydrolysis measured in the Perhydrolysis assay (Total acid detected in the hydrolysis assay-Perhydrolysis measured in the perhydrolysis assay)

The results of these experiments are provided in FIGS. 7, 10 and FIG. 11. FIG. 7 provides a graph which shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 40 minutes. FIG. 10 shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 4, 10, and 30 minutes. FIG. 11 shows the ratio of peracetic acid to acetic acid generated by various enzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10 minutes. The results obtained in these experiments indicated that M. smegmatis perhydrolase homologues exhibited a ratio above 1 in the OPDGC assays described above, while other classes of enzymes exhibited ratios significantly below 1.

Table 2-1 provides data showing the perhydrolysis activity of various homologues described herein on triacetin, as compared to the wild-type M. smegmatis perhydrolase. The results provided in Table 2-2 indicate that the perhydrolase has activity over a broad range of substrates. In addition to the results provided in these Tables, FIGS. 8 and 9 provide data showing that the perhydrolase of the present invention has broad pH and temperature range activities.

TABLE 2-1 Perhydrolysis Activity of Perhydrolase Homologues on Triacetin as Compared to M. smegmatis perhydrolase Perhydrolysis Ratio (homolog to Experiment Protein perhydrolase) A. pET26_Mlo 0.6 pET26b_Mbo 0.87 pET26_SmeII 2.1 pET26b_Stm 0.17 pLO_SmeI 0.7 Perhydrolase 1.0000 Blank 0.0660 B. pET26_S261_M2aA12 1.5 Perhydrolase 1 Blank 0.3 C. pet26 M40cD4 0.14 pet26 M44aA5 0.16 Perhydrolase 1 Blank 0.01

TABLE 2-2 Peracid Production by 1 ppm Wild-Type Perhydrolase with 29 mM H2O2 and Various Esters nmol Peracetic Acid/min 10 mM of 10 mM Ester with of Ester on 0.5% 10 mM of Polycotton Ester Neodol Ester Swatch Ethyl Acetate 5.00 Butyl Acetate 8.06 8.72 Hexyl Acetate 7.96 5.86 Octyl Acetate 8.03 0.48 Ethyl Propionate 0.90 1.43 Butyl Propionate 2.47 3.39 Hexyl Propionate 4.00 2.66 Isoamyl Acetate 7.83 17.69 Citronellyl Acetate 7.25 4.27 Citronellyl 2.85 3.21 Propionate Dodecyl Acetate 3.95 0.19 Neodol 23-3 2.25 8.77 Acetate Neodol 23-6.5 2.73 10.12 Acetate Neodol 23-9 2.97 10.20 Acetate Ethylene Glycol 13.30 Diacetate Propylene Glycol 13.17 Diacetate Triacetin 11.91 Tributyrin 0.66 2.70 Ethyl 0.49 Methoxyacetate Linalyl Acetate 0.30 Ethyl Butyrate 0.31 Ethyl Isobutyrate 0.10 Ethyl 2- 0.11 methylbutyrate Ethyl Isovalerate 0.37 Diethyl Maleate 0.75 Ethyl Glycolate 1.91

B. Typical Perhydrolase Peracid Generation Assay:

Perhydrolase is active over a wide pH and temperature range and accepts a wide range of substrates for acyl transfer. Acceptors include water (hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classical acyl transfer). For perhydrolysis measurements enzyme was incubated in the buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide. Typical substrates used to measure perhydrolysis include ethylacetate, triacetin, tributyrin, ethoxylated neodol acetate esters, and others. In addition, the wild type enzyme was found able to hydrolyze nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. In some embodiments, peracid acid and acetic acid were measured by the ABTS or HPLC assays as described below. Nitrophenylester hydrolysis is also described below.

C. ABTS Assay (One Milliliter):

This assay provides a determination of peracetic acid produced by perhydrolase. This protocol was adapted from Karst et al., Analyst, 122:567-571 [1997]). Briefly, a 100 μL aliquot of solution to be analyzed was added to 1 mL 125 mM K⁺ citrate pH 5, 1 mM ABTS, 50 μM KI. Absorbance was measured at 420 nm for highest sensitivity. However, multiple additional wavelengths were sometimes used over the broad absorption spectrum of ABTS. Calibration curves were constructed based on known peracid concentration series.

D. HPLC (Model—Agilent 1100) Determination of Perhydrolase Reaction Products:

For determination of the ratio of perhydrolysis to hydrolysis of the perhydrolase reaction, perhydrolase reaction samples were quenched by acidification to a final concentration of 0.24% methanesulfonic acid, and the products were separated by reverse phase HPLC on a Dionex OA column (cat #062903; Dionex Corporation, Sunnyvale, Calif.). The mobile phase was 100 mM NaPO4, pH 3.9 (buffer was prepared by titrating 100 mM Na₂PO4 with methanesulfonic acid to pH 3.9) run under isocratic conditions at 30° C. Detection was at 210 nm Concentrations of products were calculated by comparison of the integrated peak areas against calibration standards.

E. Nitrophenylester Hydrolysis Kinetic Assay

Enzyme and substrate were incubated in 100 mM Tris/HCl pH 8.0 (or 50 mM B(OH)₃ pH 9.5 or another buffer). Absorbance at 402 nm was monitored. In some experiments, the assay was carried out in standard 1 mL cuvettes, while in other experiments, microtiter plate wells were used. The latter method was used for the screening of mutant libraries. Enzyme concentration was determined by comparison to standard curves obtained under the same reaction conditions.

F. Para-nitrophenylcaproate Hydrolysis Assay

The pNC6 substrate solution was prepared by mixing 1 mM pNC6 (100 mM stock solution), 1 ml DMSO, 19 mls 100 mM Phosphate (pH8), and glycerol to a final concentration of 10%. To assay samples, 10 μl of the cell lysate were added to 190 μl of the substrate solution, and assayed at 405 nm for 15 minutes in a spectrophotometer. The results are presented as the average of two experiments.

G. Para-Nitrophenyl Acetate (pNA) Hydrolysis Assay

Aliquots of the lysed cell supernatant were diluted 1-100 in 100 mM phosphate buffer (pH 8). To assay the samples, 5 μl of the 1-100 diluted cell supernatant were placed into each well of a microtiter plate. Then, 195 μl of reaction buffer/substrate mix (1 mM pNA, 100 mM phosphate, pH 8, 10% glycerol) were added, and the absorbance rate at 405 nm was measured over 3 minutes (kinetics program, microtiter plate reader). The results are presented as the average of two experiments.

Example 3 Assays Including Detergent Compositions

In this Example, assay systems used to screen for superior perhydrolase activity in detergents with particular substrates are provided. These assays include those that measure peracid degradation of perhydrolase, as well as the peracid synthesis activity of the enzyme.

Materials and Methods for Peracetic Acid Formation (PAF) and Peracetic Acid Degradation (PAD) Assays

This section provides the materials and methods used to screen for a superior perhydrolases in Aria with C9E20AC ester substrate

Materials:

Aria Futur without bleach, perfume, or enzymes (P&G, Aria “C”)

C9E2OAc (P&G) 30% Hydrogen Peroxide (Sigma)

32% Peroxyacetic acid (“peracid”, PAA) (Sigma cat#) MW=76.05; 4.208M Citric Acid, anhydrous MW=192.12

Potassium Hydroxide MW=56.11

ABTS (Sigma cat# A1888) MW=548.68

Potassium Iodide MW=166.0

Potassium Phosphate, mono and di-basic

Stock Solutions:

Ariel detergent stock: Aria Futur without bleach, perfume, or enzymes (“Aria C”) was dissolved in water to 6.72 g/L. It was stirred at room temp for 30 minutes, then allowed to settle. Then, it was divided into convenient aliquots and stored at 4° C., until used. When made and used fresh, the solution was filtered, instead of settled 100 mM C9E2OAc in Ariel detergent stock: First, 30 μl C9E2OAc was added to 970 μl Aria detergent stock, using a positive displacement pipet. It was sonicated in a bath sonicator until a milky dispersion was formed (15-60 seconds). The dispersion was stable for about two hours. When used, 10 μl of dispersion per ml of reaction mix were used. 42 mM Peroxyacetic acid stock: Right before use, the Sigma 32% PAA solution was diluted 1:100 in water. Then 5.7 μl of the 42 mM stock per ml of reaction mix was added. 2 M hydrogen peroxide: One ml of 30% Sigma hydrogen peroxide was added to 3.41 ml water. This solution was prepared fresh, right before use. It was used at 10 μl per ml of reaction mix. 125 mM Citrate buffer pH 5.0: This was prepared to 24.0 grams per liter. It was made up in 800 ml, and titrated to pH 5.0 with 50% KOH. The volume was adjusted to 1 liter and stored at room temperature. 100 mM ABTS stock: This was prepared using 549 mg of ABTS in 10 ml of water. It was frozen at −80° C., in convenient aliquots in opaque Eppendorf tubes. The stock was stable indefinitely when kept frozen in the dark. ABTS will precipitate when thawed from −80° C. but goes back into solution upon mixing. In use, 10 μl of ABTS stock was used per ml of ABTS reagent. 250 mM KI: This was prepared as 415 mg in 10 ml water. It was kept at 4° C. It was diluted to 25 mM working stock, and 2 ul of working stock was used per ml of ABTS reagent. 25 mM Potassium Phosphate buffer, pH 8.0:

Method:

The night prior to performance of the assays, the plates containing lysed cells that contain perhydrolase were checked to be sure that they were frozen twice. On the day of the assay, 30 to 45 minutes were allowed for the plates to thaw. The ABTS reagent was prepared and the Multidrop (Multidrop 384 instrument, ThermoElectron) to fill the detection plates with 200 μl per well. Store the filled plates covered at room temperature in the dark until needed. Dilutions of the standards were prepared so that when 20 μl of the diluted standard were added to the 180 μl of the reaction mix, the concentration in the well was 1 ppm. Four 4 two-fold serial dilutions were prepared to a set of six standards: 1, 0.5, 0.25, 0.125, and 0.0625 ppm final concentration in the wells.

To test, 20 μl of the standards were added to the thawed 1:10 dilution plate. The reaction mixtures were prepared and the Multidrop used to fill one reaction plate for each plate to be assayed (180 μl/well). Note that the reaction mixtures are different for the PAF and PAD assays.

Peracid Hydrolysis (Peracid Degradation, PAD) Assay:

This assay measures the amount of peracetic acid remaining after a 100 minute incubation with enzyme in an Aria detergent background. The amount of peracid remaining is detected by reacting an aliquot of the reaction mixture with the ABTS detection reagent.

In this assay, 20 μl enzyme samples from the thawed 1:10 dilution plate were transferred, one column at a time with an 8 channel pipetter, into the corresponding column of the pre-filled PAD reaction plate. A timer was started as soon as transfer occurred from the first column; subsequent columns were transferred at 15 second intervals (i.e., the last column was finished 2 min 45 sec. after starting the first one). The plate was mixed for 30 seconds on the thermomixer (750 rpm, to avoid splashing). The plate was then transferred to a humidified chamber at 25° C. The plate was incubated for a total of 100 minutes from the time the first column of enzyme was added. At 100 minutes incubation, the reaction plate was removed from the incubator. Then, 20 ul aliquots of the reaction mixture were transferred to an ABTS reagent plate, in the same order and with the same 15 second time interval that the enzyme samples were originally added to the reaction plate. The ABTS plate was allowed to sit at room temperature for three minutes after the last column of reaction mixture was added. The plate was then read on the spectrophotometric plate reader at 420 and 740 nm

Perhydrolysis (Peracid Formation, PAF) Assay

Multidrop Optimized Protocol: Screening for a Superior Perhydrolysis in Ariel with C9E2OAC Ester Substrate

The same materials and stock solutions described above for PAD were used in these experiments, as indicated below.

Method:

The methods were designed to assay 20 μl aliquots from a 1:100 dilution plate. The 20 μl 1:100 dilution assay plates were produced during the process of obtaining the protein concentrations and were stored at −80° C. The plates were thawed for about 30 to 45 minutes before use. Dilutions of the S54V standards were prepared, so that when 2 μl of the diluted standard are added to the 20 μl of the 1:100 diluted cell lysate, the concentration in the well was 0.1 ppm. Four two-fold serial dilutions were prepared to produced a set of six standards: 0.1, 0.05, 0.025, 0.0125, and 0.00625 ppm final concentration in the wells. Then, 2 ul of the standards were added to the thawed 20 ul 1:100 dilution assay plates in the wells indicated.

Perhydrolysis (Peracid Formation, PAF) Assay:

This assay measures the amount of peroxyacetic acid that is produced in 10 minutes from the C9E2OAc substrate in an Aria detergent background. The amount of peracid formed is detected after 10 minutes by reacting an aliquot of the reaction mixture with the ABTS detection reagent.

The Multidrop was used to deliver 180 μl/well of the PAF reaction mix to the prepared 1:100 dilution plate. The timer was started and the reaction plate was placed on the thermomixer, with the temperature set at 25° C. The plate was covered and the solutions mixed for 30 seconds at 750 rpm. The plate was then allowed to rest on the thermomixer without mixing, for a total of 10 minutes from the time the reaction mix was added. At 10 minutes, the Multidrop was used to add 20 μl/well of the 10×ABTS reagent. The 10× reagent was a milky suspension. The thermomixer was used to briefly shake the plate. The ABTS reagent quickly went into solution. The plate was allowed to sit at room temperature for three minutes after the ABTS reagent was added. The plate was then read on the spectrophotometric plate reader at 420 nm

Example 4 Cloning of Mycobacterium smegmatis Perhydrolase

In this Example, the cloning of M. smegmatis perhydrolase is described. An enzyme with acyltransferase activity was purified from Corynebacterium oxydans (now Mycobacterium parafortuitum ATCC19686). Two peptide sequences were obtained from the purified protein. One peptide was determined by Edman degradation from cyanogen bromide cleavage of the purified enzyme using methods known in the art. The sequence of this peptide was determined to be KVPFFDAGSVISTDGVDGI (SEQ ID NO:3). The second peptide was analyzed using N-terminal sequencing and was found to have the GTRRILSFGDSLTWGWIPV (SEQ ID NO:4). A BLAST search against the TIGR unfinished genome database identified a sequence of potential interest in Mycobacterium smegmatis, which is shown below:

(SEQ ID NO: 2) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVI EEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKA YFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPH PWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHF TEANNRDLGVALAEQVRSLL.

The corresponding DNA sequence of the gene is:

(SEQ ID NO: 1) 5′-ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCT GGGTCCCCGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGT GCGCTGGACCGGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTG ATCGAGGAGGGACTGAGCGCGCGCACCACCAACATCGACGACCCCACCG ATCCGCGGCTCAACGGCGCGAGCTACCTGCCGTCGTGCCTCGCGACGCA CCTGCCGCTCGACCTGGTGATCATCATGCTGGGCACCAACGACACCAAG GCCTACTTCCGGCGCACCCCGCTCGACATCGCGCTGGGCATGTCGGTGC TCGTCACGCAGGTGCTCACCAGCGCGGGCGGCGTCGGCACCACGTACCC GGCACCCAAGGTGCTGGTGGTCTCGCCGCCACCGCTGGCGCCCATGCCG CACCCCTGGTTCCAGTTGATCTTCGAGGGCGGCGAGCAGAAGACCACTG AGCTCGCCCGCGTGTACAGCGCGCTCGCGTCGTTCATGAAGGTGCCGTT CTTCGACGCGGGTTCGGTGATCAGCACCGACGGCGTCGACGGAATCCAC TTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTCGCGGAACAGG TGCGGAGCCTGCTGTAA-3′

Primers were designed based on the gene sequence to amplify and clone the gene. The primers used for amplification were:

MsRBSF: (SEQ ID NO: 5) 5′-CTAACAGGAGGAATTAACCATGGCCAAGCGAATTCTGTGTTTCGGT GATTCCCTGACCT-3′ MspetBamR: (SEQ ID NO: 6) 5′-GCGCGCGGATCCGCGCGCTTACAGCAGGCTCCGCACCTGTTCCGCG AGGGCCACCCCGA-3′

The amplification of the gene was done by PCR using Taq DNA polymerase (Roche) per the manufacturer's instructions, with approximately 500 ng of chromosomal DNA from Mycobacterium smegmatis as the template DNA and the addition of 1% DMSO to the PCR reaction mix. Thirty picomoles of each of the primers MsRBSF and MspetBamR were added to the mix. The amplification cycle was: 30 cycles of (95° C. for 1 min, 55° C. for 1 min, 72° C. for 1 min).

The fragments obtained from the PCR reaction were separated on a 1.2% agarose gel and a single band of the expected size of 651 bp (coding sequence and stop codon) was identified. This band was cloned directly into the pCR2.1 TOPO cloning vector (Invitrogen) and transformed into E. coli Top 10 cells (Invitrogen) with selection on L agar (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 20 g/l agar) containing 100 micrograms/ml carbenicillin and X-gal (20 micrograms/ml, Sigma-Aldrich) for blue/white selection and incubated overnight at 37° C. Five white colonies were analyzed for the presence of the PCR fragment. Each colony was used to inoculate 5 mls of L broth (L agar without the addition of agar) containing 100 micrograms/ml carbenicillin and the cultures were grown overnight at 37° C. with shaking at 200 rpm. Plasmid DNA was purified from the cultures using the Quikspin kit (Qiagen). The presence of the correct fragment was determined by restriction enzyme digest with EcoR1 to release the fragment, and sequencing using primers supplied by the pCR2.1 manufacturer (Invitrogen). The correct plasmid was designated pMSATNcoI (See, FIG. 12, for the map of this plasmid)). The sequence of this plasmid is provided below

(SEQ ID NO: 13) agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatt aatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagc gcaacgcaattaatgtgagttagctcactcattaggcaccccaggctt tacactttatgcttccggctcgtatgttgtgtggaattgtgagcggat aacaatttcacacaggaaacagctatgaccatgattacgccaagctat ttaggtgacactatagaatactcaagctatgcatcaagcttggtaccg agctcggatccactagtaacggccgccagtgtgctggaattcgccctt ctaacaggaggaattaaccatggccaagcgaattctgtgtttcggtga ttccctgacctggggctgggtccccgtcgaagacggggcacccaccga gcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagct cggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccac caacatcgacgaccccaccgatccgcggctcaacggcgcgagctacct gccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcat gctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcga catcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgc gggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctc gccgccaccgctggcgcccatgccgcacccctggttccagttgatctt cgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgc gctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgat cagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcg cgatctcggggtggccctcgcggaacaggtgcagagcctgctgtaaaa gggcgaattctgcagatatccatcacactggcggccgctcgagcatgc atctagagggcccaattcgccctatagtgagtcgtattacaattcact ggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttaccca acttaatcgccttgcagcacatccccctttcgccagctggcgtaatag cgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctata cgtacggcagtttaaggtttacacctataaaagagagagccgttatcg tctgtttgtggatgtacagagtgatattattgacacgccggggcgacg gatggtgatccccctggccagtgcacgtctgctgtcagataaagtctc ccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcat gatgaccaccgatatggccagtgtgccggtctccgttatcggggaaga agtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaa cctgatgttctggggaatataaatgtcaggcatgagattatcaaaaag gatcttcacctagatccttttcacgtagaaagccagtccgcagaaacg gtgctgaccccggatgaatgtcagctactgggctatctggacaaggga aaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatg gcgatagctagactgggcggttttatggacagcaagcgaaccggaatt gccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaa gctggatggctttctcgccgccaaggatctgatggcgcaggggatcaa gctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaag atggattgcacgcaggttctccggccgcttgggtggagaggctattcg gctatgactgggcacaacagacaatcggctgctctgatgccgccgtgt tccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacc tgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgt ggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtca ctgaagcgggaagggactggctgctattgggcgaagtgccggggcagg atctcctgtcatctcaccttgctcctgccgagaaagtatccatcatgg ctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccat tcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatgg aagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggc tcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacg gcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatca tggtggaaaatggccgcttttctggattcatcgactgtggccggctgg gtgtggcggaccgctatcaggacatagcgttggctacccgtgatattg ctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacg gtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttg acgagttcttctgaattattaacgcttacaatttcctgatgcggtatt ttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcac ttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgct tcaataatagcacgtgaggagggccaccatggccaagttgaccagtgc cgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctg gaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgc cggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccagga ccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcct ggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccg ggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggg gcgggagttcgccctcgcgacccggccggcaactgcgtgcacttcgtg gccgaggagcaggactgacacgtgctaaaacttcatttttaatttaaa aggatctaggtgaagatcctttttgataatctcatgaccaaaatccct taacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatc aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttg caaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaa gagctaccaactctttttccgaaggtaactggcttcagcagagcgcag ataccaaatactgtccttctagtgtagccgtagttaggccaccacttc aagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta ccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagttaccggataaggcgcagcggtcgggctgaacgggg ggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactg agatacctacagcgtgagctatgagaaagcgccacgcttcccgaaggg agaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcg tcaggggggcggagcctatggaaaaacgccagcaacgcggccttttta cggttcctgggcttttgctggccttttgctcacatgttctttcctgcg ttatcccctgattctgtggataaccgtattaccgcctttgagtgagct gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagc gaggaagcggaag

Construction of Perhydrolase T7 Expression Plasmid

The primer pair used to create pMSATNco1 was also used to create an NcoI site (CCATGG) in which the ATG is the start codon of the acyltransferase gene and a BamH1 (GGATCC) just after the TAA stop codon. The plasmid pMSATNco1 was digested with NcoI/BamH1 as recommended by the manufacturer (Roche) and the 658 bp fragment containing the perhydrolase gene was purified using standard procedures known in the art (e.g., Sambrook et al.). The fragment was ligated using standard procedures known in the art (e.g., Sambrook et al.) into the T7 promoter expression plasmid, pET16b (Novagen), also digested with NcollBamH1. The ligation reaction was transformed by standard procedures into E. coli Top 10 cells (Invitrogen) and selected on L agar containing 100 micrograms/ml carbenicillin overnight at 37° C. Ten colonies were picked from the several transformants and used to inoculate 5 ml of LB containing 100 micrograms/ml carbenicillin. Cultures were grown overnight at 37° C. with shaking at 200 rpm. Plasmid DNA was purified from the cultures using the Qiagen Quikspin kit (Qiagen). The presence of the correct fragment was determined by restriction enzyme digest with NcoI/BamH1 as directed by the manufacturer. The correct plasmid was designated pMSATNcoI-1 (See, FIG. 13, for the map of this plasmid). In this Figure, the following elements are indicated—Lac I: gene encoding the Lad repressor protein, located at bp1455-2534, ori: plasmid origin of replication at by 4471, bla: The β-lactamase gene located at by 6089-5232; T7 promoter: located at bp1068-1052; T7 terminator: located at by 259-213, per: the M. smegmatis perhydrolase gene located at 981-334. The sequence of this plasmid is provided below:

(SEQ ID NO: 131) ttctcatgtttgacagcttatcatcgataagctttaatgcggtagttt atcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaa caatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtagg cataggcttggttatgccggtactgccgggcctcttgcgggatatccg gatatagttcctcctttcagcaaaaaacccctcaagacccgtttagag gccccaaggggttatgctagttattgctcagcggtggcagcagccaac tcagcttcctttcgggctttgttagcagccggatccgcgcgcttacag caggctccgcacctgttccgcgagggccaccccgagatcgcgattgtt ggcctcggtgaagtggattccgtcgacgccgtcggtgctgatcaccga acccgcgtcgaagaacggcaccttcatgaacgacgcgagcgcgctgta cacgcgggcgagctcagtggtcttctgctcgccgccctcgaagatcaa ctggaaccaggggtgcggcatgggcgccagcggtggcggcgagaccac cagcaccttgggtgccgggtacgtggtgccgacgccgcccgcgctggt gagcacctgcgtgacgagcaccgacatgcccagcgcgatgtcgagcgg ggtgcgccggaagtaggccttggtgtcgttggtgcccagcatgatgat caccaggtcgagcggcaggtgcgtcgcgaggcacgacggcaggtagct cgcgccgttgagccgcggatcggtggggtcgtcgatgttggtggtgcg cgcgctcagtccctcctcgatcacctcgaagtccgctccgagctgctg ggccagcacaccggtccagcgcacgtcgggggcgaaccgctcggtggg tgccccgtcttcgacggggacccagccccaggtcagggaatcaccgaa acacagaattcgcttggccatggtatatctccttcttaaagttaaaca aaattatttctagaggggaattgttatccgctcacaattcccctatag tgagtcgtattaatttcgcgggatcgagatctcgatcctctacgccgg acgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgc ctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgg gctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggc cgggggactgttgggcgccatctccttgcatgcaccattccttgcggc ggcggtgctcaacggcctcaacctactactgggctgcttcctaatgca ggagtcgcataagggagagcgtcgagatcccggacaccatcgaatggc gcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaat tcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagt atgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggcca gccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcgg agctgaattacattcccaaccgcgtggcacaacaactggcgggcaaac agtcgttgctgattggcgttgccacctccagtctggccctgcacgcgc cgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtg ccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgta aagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatca ttaactatccgctggatgaccaggatgccattgctgtggaagctgcct gcactaatgttccggcgttatttcttgatgtctctgaccagacaccca tcaacagtattattttctcccatgaagacggtacgcgactgggcgtgg agcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcc cattaagttctgtctcggcgcgtctgcgtctggctggctggcataaat atctcactcgcaatcaaattcagccgatagcggaacgggaaggcgact ggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagg gcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgg gcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggata tctcggtagtgggatacgacgataccgaagacagctcatgttatatcc cgccgttaaccaccatcaaacaggattttcgcctgctggggcaaacca gcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggca atcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgc ccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgc agctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtaagttagctcactcattaggcaccgggatctcgaccg atgcccttgagagccttcaacccagtcagctccttccggtgggcgcgg ggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaa ctcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggac cgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattc ggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccacc aaacgtttcggcgagaagcaggccattatcgccggcatggcggccgac gcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggcc ttccccattatgattcttctcgcttccggcggcatcgggatgcccgcg ttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacag cttcaaggatcgctcgcggctcttaccagcctaacttcgatcactgga ccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaac gggttggcatggattgtaggcgccgccctataccttgtctgcctcccc gcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaa gccggcggcacctcgctaacggattcaccactccaagaattggagcca atcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcaga acatatccatcgcgtccgccatctccagcagccgcacgcggcgcatct cgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgt cgttgaggacccggctaggctggcggggttgccttactggttagcaga atgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaa cgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgttt cgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttcc ggatctgcatcgcaggatgctgctggctaccctgtggaacacctacat ctgtattaacgaagcgctggcattgaccctgagtgatttttctctggt cccgccgcatccataccgccagttgtttaccctcacaacgttccagta accgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctc gtttcatcggtatcattacccccatgaacagaaatcccccttacacgg aggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgct ttatcagaagccagacattaacgcttctggagaaactcaacgagctgg acgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctg atgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaa acctctgacacatgcagctcccggagacggtcacagcttgtctgtaag cggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttg gcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggag tgtatactggcttaactatgcggcatcagagcagattgtactgagagt gcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaa taccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgc tcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggta atacggttatccacagaatcaggggataacgcaggaaagaacatgtga gcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctg gcgtttttccataggctccgcccccctgacgagcatcacaaaaatcga cgctcaagtcagaggtggcgaaacccgacaggactataaagataccag gcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcg ctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgc tgcgccttatccggtaactatcgtcttgagtccaacccggtaagacac gacttatcgccactggcagcagccactggtaacaggattagcagagcg aggtatgtaggcggtgctacagagttcttgaagtggtggcctaactac ggctacactagaaggacagtatttggtatctgcgctctgctgaagcca gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacc accgctggtagcggtggtttttttgtttgcaagcagcagattacgcgc agaaaaaaaggatctcaagaagatcctttgatcttttctacggggtct gacgctcagtggaacgaaaactcacgttaagggattttggtcatgaga ttatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagt tttaaatcaatctaaagtatatatgagtaaacttggtctgacagttac caatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgt tcatccatagttgcctgactccccgtcgtgtagataactacgatacgg gagggcttaccatctggccccagtgctgcaatgataccgcgagaccca cgctcaccggctccagatttatcagcaataaaccagccagccggaagg gccgagcgcagaagtggtcctgcaactttatccgcctccatccagtct attaattgttgccgggaagctagagtaagtagttcgccagttaatagt ttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcg tcgtttggtatggcttcattcagctccggttcccaacgatcaaggcga gttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggt cctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatg gttatggcagcactgcataattctcttactgtcatgccatccgtaaga tgcttttctgtgactggtgagtactcaaccaagtcattctgagaatag tgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataat accgcgccacatagcagaactttaaaagtgctcatcattggaaaacgt tcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagt tcgatgtaacccactcgtgcacccaactgatcttcagcatcttttact ttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgca aaaaagggaataagggcgacacggaaatgttgaatactcatactcttc ctttttcaatattattgaagcatttatcagggttattgtctcatgagc ggatacatatttgaatgtatttagaaaaataaacaaataggggttccg cgcacatttccccgaaaagtgccacctgacgtctaagaaaccattatt atcatgacattaacctataaaaataggcgtatcacgaggccctttcgt cttcaagaa

This plasmid was transformed into the E. coli strain BL21(λDE3)pLysS (Novagen), which contains the gene encoding the T7 RNA polymerase, with selection on LA containing 100 micrograms/ml carbenicillin. Cells were grown overnight at 37° C. One transformant was selected and the strain was designated MSATNco1.

Production of Perhydrolase in MSATNco1-1

Production of perhydrolase was done in cell culture. For example, 5 ml of LB with carbenicillin at a concentration of 100 micrograms/ml was inoculated with a single colony of MSATNco1 and grown overnight at 37° C. with shaking at 200 rpm. This culture was used to inoculate 100 ml of LB with carbenicillin at a concentration of 100 micrograms/ml (in a 250 ml baffled flask) to an OD₆₀₀ of 0.1. The cultures were grown at 30° C. with shaking at 200 rpm until they reached an OD₆₀₀ of 0.4. The expression of the perhydrolase gene was then induced by the addition of 100 micromolar IPTG and the incubation continued overnight. Cultures were harvested by centrifugation (10 min at 7000 rpm, Sorvall SS34 rotor), the supernatant was removed and the pellets washed in 50 mM KPO4, pH 6.8. The cells were centrifuged again, the supernatants removed and the wet weight of the cells was determined. The cells were resuspended in 100 mM KPO4 in a volume that was 4× the wet weight. The resuspended cells were frozen at −70° C. The cells were thawed and lysed in a French Pressure cell using standard procedures known in the art. The purification steps and assessment methods are provided in Example 1. FIG. 6 provides a purification table showing the enzyme activity of the perhydrolase of the present invention through various steps in the purification process.

M. smegmatis Perhydrolase is in an Operon

In additional experiments, it was determined that the M. smegmatis perhydrolase is part of an operon. The gene (phd) is the first gene in an operon that contains at least 2 genes, including phd, that are separated by 10 bp (GGCTGGGGGC [SEQ ID NO:7]) not including the TAA stop codon of phd. It is also possible that there are three genes in the operon, with the third being either 48 bp or 61 bp to the next ORF (open reading frame). The latter two candidate genes have no significant homology to proteins in the database.

A putative promoter was identified for M. smegmatis phd operon, TTGGGC (−35) SP (18) CCAGAT by sequence analysis and comparison with known M. smegmatis promoters (See e.g., Salazar et al., Microbiol., 149:773-784 [2003]). It is not intended that the present invention be limited to any particular promoter and/or construct design, as it is contemplated that other promoters and construct designs will find use in the present invention.

The second gene in the phd operon encodes a protein (putative PBP-3) with the sequence:

(SEQ ID NO: 9) mhlrpaltwllvvglfisvvgcssspdpadrfsafaealgrkdaaaaaaq tsdpaaaeaaitamlagmgdaanysvaaepeegddagatlkytwtwgegr dfgydttataaksgddwlitwsptylhrdltpdlrfqysedselqtpvld rtgqplmtwqtygvitverahpesaaplaallapfdpattesvtaqlnsa ddrvtvmklreddlgqvrdqlaqipgvtvreqgelltadrqlsspaisgl delwhdritanagwsvylvdadgapaqqltstppkdtgpvrttldlrmql laqqavaketrpavvvaisgstggilaaaqnpaadpqgaiafsglyppgs tfktittaaaldaglatpdtpvacpgeltienrtipnddnfdlgtvplss afshscntsmaalsdelppnaltdmakdfgigvdfmvpglttvtgrvpna dnaaqrvengigqgtvtvspfglavaeaslahgstilptlvdgekttadt psvplppnitdalrammrgtvtegtatalsdipdlggktgtaefgdnths hgwfagiagdiafatlvvggdssapavaisgdflrpalag

The corresponding DNA sequence of the gene encoding the putative PBP-3:

(SEQ ID NO: 8) atgcacttacgtcccgctctgacgtggctcctggttgtcggtctgttcatatcggtcgtcggatgttcgtcgtccccggatccggccg accggttctcggcgttcgccgaggcgctgggccgcaaggatgcggccgcggcggccgcccagaccagcgatccggcggcc gcggaggcggccatcaccgcgatgctggccgggatgggcgacgccgcgaacgtctcggtggccgccgaacccgaggaagg cgacgacgcgggcgcgacgctgaagtacacgtggacctggggtgagggccgcgacttcggctacgacaccaccgcgacggc ggccaaatccggtgacgactggctgatcacctggtcccccaccgtgttgcaccgcgacctcaccccggatctgcgcttccagtac agcgaggacagcgaattgcagaccccggtgctcgaccgcaccggccagccgttgatgacatggcagaccgtcggtgtcatcac tgtcgaacgcgcacatccggagtcggccgcaccgctcgccgccctgctggcgcccttcgatccgaccaccaccaccgaatcgg tcaccgcacaactcaattcgacgaccgatgaccgcgtgacggtgatgaagctgcgcgaggacgatctgggtcaggtgcgcgat cagctcgcgcagatccccggcgtgaccgtgcgtgagcagggtgagctgctcaccgccgaccggcagctgtcctcgcccgccat cagcggcctggacgagctgtggcacgaccggatcaccgccaacgcgggctggtcggtgtacctggtcgacgccgacggtgca cccgcacaacagctcacgtccacgccgcccaaggacaccgggcccgtgcgcaccacgctggacctgcgcatgcaactgctcg cgcagcaggccgtggccaaggagacccgcccggccgtggtggtcgcgatctccggatcgaccgggggcatcctggccgccg cacagaacccggccgccgatccgcaaggtgcgatcgcgttttcgggcctgtacccgccggggtcgacgttcaagaccatcacc acggcggcagccctcgacgcgggcctggccaccccggacacaccggtggcctgcccgggtgagctcaccatcgagaaccgc acgatccccaacgacgacaacttcgacctgggcaccgtgccgttgtcgtcggcgttctcgcactcctgcaacaccagcatggcc gccctgtccgacgagctgccgcccaacgcactgaccgacatggcaaaggacttcgggatcggcgtcgacttcatggtgcccgg cctgaccaccgtgaccggccgtgtccccaacgccgacaacgccgcccagcgtgtcgagaacggcatcggccagggcaccgt gaccgtcagcccgttcggcctcgccgtcgccgaggccagcctggcgcacggttcgacgatcctgccgacgctggtcgacggc gagaagaccacggccgacaccccgtcggtgccgttgccgcccaacatcaccgacgcgctgcgcgcgatgatgcgcggaacg gtcaccgagggcacggccaccgcgttgagcgacatccccgacctgggcggcaagaccggcacggcggaattcggcgacaac acgcactcgcacggctggttcgcgggcatcgcgggcgacatcgcgttcgcgacgctggtggtcggcggcgactcgtcggcac cggccgtcgcgatctcaggagacttcctgcgccccgcgctcgccggctag.

A standard BLAST search against the protein database identified homology with several penicillin binding proteins, class 3 (PBP-3). By sequence alignment and comparison to literature (e.g., Goffin and Ghysen, Microbiol. Mol. Biol. Rev., 66:702-38 [2002]) the PBP was found to contain the required bar codes (conserved protein sequences that define a class of proteins) to place it in the SxxK superfamily of acyl transferases, with a C-terminal domain acyl transferase and an N-terminal domain of unknown function, but with homology to the Pen^(r) (i.e., penicillin resistant) protein fusions of class B-like II and III. This penicillin binding protein acyl transferase domain does not share significant homology with the perhydrolase of the present invention, although it does share homology with Co-A dependent acyl transferases known in the art. The amino acid sequence is provided below.

(SEQ ID NO: 10) MHLRPALTWLLVVGLFISVVGCSSSPDPADRFSAFAEALGRKDAAAAAAQ TSDPAAAEAAITAMLAGMGDAANVSVAAEPEEGDDAGATLKYTWTWGEGR DFGYDTTATAAKSGDDWLITWSPTVLHRDLTPDLRFQYSEDSELQTPVLD RTGQPLMTWQTVGVITVERAHPESAAPLAALLAPFDPTTTTESVTAQLNS TTDDRVTVMKLREDDLGQVRDQLAQIPGVTVREQGELLTADRQLSSPAIS GLDELWHDRITANAGWSVYLVDADGAPAQQLTSTPPKDTGPVRTTLDLRM QLLAQQAVAKETRPAVVVAISGSTGGILAAAQNPAADPQGAIAFSGLYPP GSTFKTITTAAALDAGLATPDTPVACPGELTIENRTIPNDDNFDLGTVPL SSAFSHSCNTSMAALSDELPPNALTDMAKDFGIGVDFMVPGLTTVTGRVP NADNAAQRVENGIGQGTVTVSPFGLAVAEASLAHGSTILPTLVDGEKTTA DTPSVPLPPNITDALRAMMRGTVTEGTATALSDIPDLGGKTGTAEFGDNT HSHGWFAGIAGDIAFATLVVGGDSSAPAVAISGDFLRPALAG

The family-identifying bar codes provided in the above review were: (19) V (20) GA (140) PVxDRTG (142) TxDx3Q (22) TGGxLAx4 PaxDP (13) SxxK (51) SCN (131) KTG (50) marked in bold letters in the above sequence. The letters represent the amino acid sequence defining the bar code; the numbers in brackets are the intervening number of amino acids between the particular bar codes; “x” represents any amino acid, (i.e., the amino acids are not conserved within the bar code but the number of amino acids (e.g., x3 corresponding to 3 intervening amino acids) is conserved). Based on these results and other data, as described herein, it is clear that the perhydrolase of the present invention represents a unique enzyme class.

Example 5 Expression of the Perhydrolase in P. citrea

In this Example, methods used to express the perhydrolase in P. citrea are described. The plasmid pMSATNcoI was transformed into P. citrea by electroporation using the method essentially as known in the art (See e.g., Sambrook et al., supra) except that all cultures and recovery were done at 30° C. The transformants were plated on L agar+carbenicillin (200 μg/ml) and incubated overnight at 30° C. Three transformants were picked for analysis. Each colony was used to inoculate a 30 ml culture of LB+carbenicillin (200 μg/ml) and grown overnight at 30° C. with shaking at 200 rpm. The cells were pelleted by centrifugation, washed one time in 50 mM phosphate buffer pH 7.2, and finally resuspended in 4× the wet cell weight of 100 mM phosphate buffer pH 8.0. The cells were lysed by treatment with lysozyme (2 μl of a 10 mg/ml solution per one ml of P. citrea culture) at 37° C. for one hour. The cell debris was pelleted at 13,000 rpm in a microfuge for 5 min. The resulting supernatant was used for further analysis in SDS-PAGE and Western blots, as well as assays for enzyme activity.

SDS-PAGE analysis was carried out as known in the art (See e.g., Sambrook et al., supra) on the supernatants. Detection of the perhydrolase protein by Western blot was done using an anti-perhydrolase polyclonal anti-sera (prepared from purified perhydrolase protein by Covance). The blot was developed as per manufacturer's suggestions using the ECL plus kit (Amersham).

The enzymatic activity of the expressed perhydrolase was detected by the pNB (para-nitrophenylbutyrate) assay as described in Example 1, herein. The results are provided in the

TABLE 5-1 Enzymatic Activity of Perhydrolase Expressed by P. citrea Concentration Clone OD405 Rate (mg/liter) P. citreal 3.1129 0.47948 7.1922 pMSATNcoI Control (P. citrea) 2.6187 −9.8312 0

The SDS-PAGE and Western blot results, as well as the assay results indicated that the perhydrolase is expressed by P. citrea and is active.

Example 6 Expression of the Perhydrolase in Bacillus subtilis

The perhydrolase was expressed intracellularly in B. subtilis. A variety of promoters find use in this embodiment, including but not limited to pSPAC, pAprE, pAmyE, pVeg, pHpaII. In some embodiments, the construct is present on a replicating plasmid (e.g., pBH1), while in other embodiments, it is integrated into the chromosome in one or more copies. Examples of sites for integration include, but are not limited to the aprE, the amyE, the veg or the pps regions. Indeed, it is contemplated that other sites known to those skilled in the art will find use in the present invention.

A. Intracellular Expression of the Perhydrolase in Bacillus subtilis From a Replicating Plasmid

B. subtilis expresses a lipaseesterase encoded by the gene pnbA that hydrolyzes the pNB substrate used to detect activity of the perhydrolase. To identify B. subtilis strains expressing the perhydrolase after transformation with replicating or integrating plasmids the pnbA gene (the entire coding sequence) was first deleted from the desired host using the loxP cassette deletion method described in WO 03/083125, herein incorporated by reference. It is also noted that other strains of Bacillus may contain one or more lipases/esterases capable of hydrolyzing the pNB or other substrate used as an indicator for perhydrolase activity. In some embodiments, for optimal expression and/or activity detection it is necessary to delete one or more of the lipases/esterases from the hosts. The Bacillus subtilis strain used in this Example has the genotype Bacillus subtilis comK pnbA (pnbA loxP-spec, aprE, nprE, degUHy32, oppA, spoIIE3501 and will be referred to as “B. subtilis pnbA” (See e.g., WO 03/083125, supra).

In these experiments, a consensus Bacillus ribosome binding site (RBS) was used.

It is not intended that the consensus RBS be the only sequence used for expression, as a non-consensus RBS also finds use in the present invention. The RBS of pMSATNcoI (See, Example 4) was changed to a Bacillus consensus RBS from the 16S rRNA (5′-ATAAGGAGGTGATC-3′ ISEQ ID NO:1321) of B. subtilis and a HindIII site was added to the 5′ end of the RBS by PCR using a primer (502rbsforward primer) containing the desired changes. The reaction was carried out using an MJ Research PCR machine with 30 cycles of (1 min at 95° C., 1 min at 55° C., and 1 min at 72° C.). Template DNA (pMSATrbs) was added to a 50 μl reaction (10 ng) and 10 picomoles of each primer were used.

The PCR-generated phd cassette was cloned into the PCR cloning vector, pCR-Script CM (Stratagene) and transformed into E. coli Top10 cells (Invitrogen) to make pAH502R. The complete sequence of this plasmid is provided below.

(SEQ ID NO: 133) ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccg aaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtcca ctattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcacc ctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagc ggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcg caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgat taagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccggggg atccgcccaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcggtgattccctgacctggggc tgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagct cggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctca acggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgac accaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgc gggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccct ggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatg aaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcg cgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccccgggaagcttgcatgggctagagcg gccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagc tgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggt gcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagct gcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgc tgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggat aacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagat accaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgccttt ctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagct gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttc ttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagatta cgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgt taagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttcgaccgaataaatacctgtgacggaag atcacttcgcagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaacttttggcgaaaatgagac gttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttgtcgag attttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgta aagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggccttttta aagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccgga attacgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaa ctgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcg tgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgag tttcaccagttttgatttaaacgtggccaatatggacaacttcttcgccccgttttcaccatgggcaaatattatacgca aggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgctta atgaattacaacagtactgcgatgagtggcagggcggggcgtaatttttttaaggcagttattggtgcccttaaacgcct ggttgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagcaaattcgacccggtcgtcggttca gggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccgtgtg cttctcaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcctttttttctgat caaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatg taacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagca tttatcaagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcac atttccccgaaaagtgccac

Transformants were selected on L agar containing 100 μg/ml carbenicillin. The construct was confirmed by sequencing and biochemical assays (e.g., pNB activity assay)

Primer set for pAH502R construction:

502rbsForward primer: (SEQ ID NO: 134) 5′-ccaagcttaaggaggtgatctagaattccatggccaagcgaattctg tgtttcg-3′ 502Reverse Primer: (SEQ ID NO: 135) 5′-ggggatccttttacagcaggctccgcacct-3′

The HindIII-RBS-phd-BamH I DNA fragment from pAH502R was cloned into the pSPAC containing vector, pMUTIN4 (See, Vagner et al., Microbiol., 144, 3097-3104 [1998]) creating the construct pAH503. The complete sequence of pAH503 is provided below:

(SEQ ID NO: 136) ataattctacacagcccagtccagactattcggcactgaaattatgggtgaagtggtcaagacctcactaggcaccttaa aaatagcgcaccctgaagaagatttatttgaggtagcccttgcctacctagcttccaagaaagatatcctaacagcacaa gagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattttgcaaaaagttgttgactt tatctacaaggtgtggcataatgtgtggaattgtgagcgctcacaattaagcttaaggaggtgatctagaattccatggc caagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcg cccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcg cgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacct gccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgc tgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctg gtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccac tgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccg acggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctg ctgtaaaaggatccccagcttgttgatacactaatgcttttatatagggaaaaggtggtgaactactgtggaagttactg acgtaagattacgggtcgaccgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagc tggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctg gtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaa actggcagatgcacggttacgatgcgcccatctacaccaacgtaacctatcccattacggtcaatccgccgtttgttccc acggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattat ttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgt ctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgttggagtgacggcagt tatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaat cagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcg agttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggc ggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggag cgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcg atgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaac cgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaa caactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatg tggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcg atgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatc aggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcg gcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtg ccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgat gggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctggg actgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacg ccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaaca ccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgata acgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaa ggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgca accgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtga cgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgt tggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcga tcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaac gctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctg attacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtgg tcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctgg cgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgtttt gaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcg cgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatgg aaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggc gacgactcctggagcccgtcagtatcggcggaattacagctgagcgccggtcgctaccattaccagttggtctggtgtca aaaataataataaccgggcaggccatgtctgcccgtatttcgcgtaaggaaatccattatgtactatttcaagctaattc cggtggaaacgaggtcatcatttccttccgaaaaaacggttgcatttaaatcttacatatgtaatactttcaaagactac atttgtaagatttgatgtttgagtcggctgaaagatcgtacgtaccaattattgtttcgtgattgttcaagccataacac tgtagggatagtggaaagagtgcttcatctggttacgatcaatcaaatattcaaacggagggagacgattttgatgaaac cagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccac gtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaact ggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcga ttaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcg gcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgt ggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctccc atgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccatta agttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacg ggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgc tggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcg gtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgct ggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctg gcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttaggcatcgcatcctgtctcgc gtcgtcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgg gagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgata gcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgc acagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcgg ctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacat gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctg acgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgt ggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcca ctggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaa ctacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga tctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcat gagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagt aaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagtt gcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgaga cccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactt tatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgtt gttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaag gcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttgg ccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacggga taataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatct taccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtt tctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatact cttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaa ataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacatta acctataaaaataggcgtatcacgaggccctttcgtcttcaagaattgatcctctagcacaaaagaaaaacgaaatgata caccaatcagtgcaaaaaaagatataatgggagataagacggttcgtgttcgtgctgacttgcaccatatcataaaaatc gaaacagcaaagaatggcggaaacgtaaaagaagttatggaaataagacttagaagcaaacttaagagtgtgttgatagt gcagtatcttaaaattttgtataataggaattgaagttaaattagatgctaaaaatttgtaattaagaaggagtgattac atgaacaaaaatataaaatattctcaaaactttttaacgagtgaaaaagtactcaaccaaataataaaacaattgaattt aaaagaaaccgataccgtttacgaaattggaacaggtaaagggcatttaacgacgaaactggctaaaataagtaaacagg taacgtctattgaattagacagtcatctattcaacttatcgtcagaaaaattaaaactgaatactcgtgtcactttaatt caccaagatattctacagtttcaattccctaacaaacagaggtataaaattgttgggagtattccttaccatttaagcac acaaattattaaaaaagtggtttttgaaagccatgcgtctgacatctatctgattgttgaagaaggattctacaagcgta ccttggatattcaccgaacactagggttgctcttgcacactcaagtctcgattcagcaattgcttaagctgccagcggaa tgctttcatcctaaaccaaaagtaaacagtgtcttaataaaacttacccgccataccacagatgttccagataaatattg gaagctatatacgtactttgtttcaaaatgggtcaatcgagaatatcgtcaactgtttactaaaaatcagtttcatcaag caatgaaacacgccaaagtaaacaatttaagtaccgttacttatgagcaagtattgtctatttttaatagttatctatta tttaacgggaggaaataattctatgagtcgcttttgtaaatttggaaagttacacgttactaaagggaatgtagataaat tattaggtatactactgacagcttccaaggagctaaagaggtccctagactctagacccggggatctctgcagtcggatc tggtaatgactctctagcttgaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg tttgtcggtgaacgctctcctgagtaggacaaatccgccgctctagctaagcagaaggccatcctgacggatggcctttt tgcgtttctacaaactcttgttaactctagagctgcctgccgcgtttcggtgatgaagatcttcccgatgattaattaat tcagaacgctcggttgccgccgggcgttttttatgcagcaatggcaagaacgttgctctaga

The construction of pAH503 was confirmed by RFLP and pNB activity assays. The pSPAC-RBS-phd DNA cassette was isolated as a BglII/SmaI digest and then subcloned into the replicating plasmid pBH1, digested with BamH1/EcoRV (See e.g., EP 0275509) to create pAH505 (See, FIG. 14). The complete sequence of the plasmid is provided below.

(SEQ ID NO: 137) gatcttccaagatatcctaacagcacaagagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattt tgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtggaattgtgagcgctcacaattaagcttaaggaggtgatctag aattccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagc ggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgag cgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcac ctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctg ggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggt ctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcg cccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacg gaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccc atcgcatgcggtacctctagaagaagcttggagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaac ctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataa aaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagtt gctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgtt aaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaa tcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaact tctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaa agaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtca caaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaa gaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatg cgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaa gaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagt tcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacgga ttttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtg gtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccg atgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgt tgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctc caaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaag aaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttg gtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggaca accggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgctt acacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcc caaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaagga ccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgac gagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaac tttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtg gatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagta attatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatg agcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggca ttgccagtcggggatattaaaaagagtataggtttttattgcgataaactaggtttcactttggttcaccatgaagatggattcgcagtt ctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgctctcgtagtaatgattcaccggtttgtacag gtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggc attttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagctt ttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctctt gtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaag ctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtccct tgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaa aagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcaggacacacactttatgaatataaagtatagtgtg ttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttat gcagtttgtagaatgcaaaaagtgaaatcagggg

The ligation mixture for pAH505 was transformed into Bacillus subtilis pnbA. Correct transformants were verified by RFLP and sequencing of isolated plasmid DNA.

One transformant was selected for analysis (B. subtilis pnbA/pAH505). Expression of the perhydrolase in Bacillus was assayed using the pNB Activity Assay described herein, after growth of the desired strain in shake flask. The data showed that the perhydrolase was expressed in B. subtilis pnbA.

B. Intracellular Expression of the Perhydrolase in B. subtilis pnbA by Integration into the Chromosome

An additional construct useful to determine expression of the perhydrolase (act) gene integrated into the chromosome of B. subtilis pnbA involved use of the spoVG promoter, which was found to drive expression of the perhydrolase gene in a non-replicating (i.e., integrating plasmid). In some embodiments, one site of integration is the aprE region of B. subtilis, although it is intended that integration occur at any suitable site. Indeed, it is not intended that the present invention be limited to this specific site nor this specific promoter, as various other suitable sites and promoters find use in the present invention.

The configuration of the promoter/gene at the aprE locus in the chromosome of Bacillus subtilis was as follows:

pAprE-aprE first 7 codons-translation stop-pSpoVG-ATG-perhydrolase gene from second codon

The clone was constructed as described below. The primers used were:

Up5′F (SEQ ID NO: 138) caggctgcgcaactgttgggaag FuaprEAct34R (SEQ ID NO: 139) agtagttcaccaccttttccctatataaaagcattagtgtatcaatttca gatccacaattttttgcttctcactctttac FuaprEAct4F (SEQ ID NO: 140) Aattgatacactaatgcttttatatagggaaaaggtggtgaactact atggccaagcgaattctgtgtttcggtg BsmI-DnAct504R (SEQ ID NO: 141) gtgagaggcattcggatccttttacagcaggctccg

PCR fusion is a technique well known in the art, in which two or more fragments of DNA are generated either by restriction digest or by PCR amplification. The fragments have overlapping segments, usually at least 18 bases long. In the instance that two fragments are used, the 3′ end of fragment #1 has an overlapping sequence with the 5′ end of fragment #2. The two fragments are used as template in a PCR reaction in which the primer set used hybridizes to the 5′ end of fragment #1 (forward primer) and the 3′ end of fragment #2 (reverse primer). During the amplification, the two regions of overlap hybridize forming a single template from which the two primers can amplify a full length fragment, a “fusion” of fragments #1 and #2. Multiple fragments of any length can be used in such a reaction, limited only by the ability of the chosen polymerase to amplify long DNA pieces.

In the current example, the above construct was made by PCR fusion of two PCR products the above construct was made by PCR fusion of two PCR products. The first was a construct with the spoVG promoter added upstream of the phd gene. The second was the aprE promoter and first 7 codons of aprE, followed by a stop codon. Regions of 20 bp overlap were added on the 5′ and 3′ ends of the products respectively, to allow the PCR fusion reaction. The primer set FuaprEAct4F/BsmI-DnAct504R was used to amplify the perhydrolase gene from pAH505 as described above, which added the spoVG promoter sequence (contained within the primer) to the 5′ end of the gene and changed the start codon from ATG to GTG. To create the second product (pAprE plus the first 7 codons of aprE) for the fusion, the primer set Up5′F/FuaprEAct34R was used to amplify a fragment from pBSFNASally. FIG. 15 provides a map of this plasmid. The complete sequence of pBSFNASally is provided below.

(SEQ ID NO: 142) ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaat cccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactc caacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgagg tgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaa aggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacaccc gccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggc ctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgac gttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcggccgctcta gaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaa aaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctgatgt ctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatactt ttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacagg aatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgt atgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtcta ctaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaaga gtgagaagcaaaaaattgtggatcagtttgctgtttgctttagcgttaatctttacgatggcgttcggcagcacatcctctgcccaggc ggcagggaaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgagcacgatgagcgccgctaagaagaaag atgtcatttctgaaaaaggcgggaaagtgcaaaagcaattcaaatatgtagacgcagcttcagctacattaaacgaaaaagctgta aaagaattgaaaaaagacccgagcgtcgcttacgttgaagaagatcacgtagcacatgcgtacgcgcagtccgtgccttacggc gtatcacaaattaaagcccctgctctgcactctcaaggctacactggatcaaatgttaaagtagcggttatcgacagcggtatcgatt cttctcatcctgatttaaaggtagcaggcggagccagcatggttccttctgaaacaaatcctttccaagacaacaactctcacggaa ctcacgttgccggcacagttgcggctcttaataactcaatcggtgtattaggcgttgcgccaagcgcatcactttacgctgtaaaagt tctcggtgctgacggttccggccaatacagctggatcattaacggaatcgagtgggcgatcgcaaacaatatggacgttattaaca tgagcctcggcggaccttctggttctgctgctttaaaagcggcagttgataaagccgttgcatccggcgtcgtagtcgttgcggcag ccggtaacgaaggcacttccggcagctcaagcacagtgggctaccctggtaaatacccttctgtcattgcagtaggcgctgttgac agcagcaaccaaagagcatctttctcaagcgtaggacctgagcttgatgtcatggcacctggcgtatctatccaaagcacgcttcc tggaaacaaatacggcgcgttgaacggtacatcaatggcatctccgcacgttgccggagcggctgctttgattctttctaagcacc cgaactggacaaacactcaagtccgcagcagtttagaaaacaccactacaaaacttggtgattctttctactatggaaaagggctg atcaacgtacaggcggcagctcagtaaaacataaaaaaccggccttggccccgccggttttttattatttttcttcctccgcatgttca atccgctccataatcgacggatggctccctctgaaaattttaacgagaaacggcgggttgacccggctcagtcccgtaacggcca agtcctgaaacgtctcaatcgccgcttcccggtttccggtcagctcaatgccgtaacggtcggcggcgttttcctgataccgggag acggcattcgtaatcggatcctctagagtcgatttttacaagaattagctttatataatttctgtttttctaaagttttatcagctacaaaaga cagaaatgtattgcaatcttcaactaaatccatttgattctctccaatatgacgtttaataaatttctgaaatacttgatttctttgttttttct cagtatacttttccatgttataacacataaaaacaacttagttttcacaaactatgacaataaaaaaagttgctttttcccctttctatgtat gttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaaaaccagtcataccaataacttaagg gtaactagcctcgccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaac acaaaagaccacattttttaatgtggtctttattcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatt tttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattc actttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcatta tttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaatttta ccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattg ggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagag ttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctcttt ctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattca ttaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctat tcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcacta acctgccccgttagttgaagaaggtttttatattacagctccagatccatatccttctttttctgaaccgacttctcctttttcgcttctttatt ccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggtcggcttaatagctcacgctatgccga cattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcggcataaatgcgtggtctaatttttatttttaataacctt gatagcaaaaaatgccattccaatacaaaaccacatacctataatcgaccggaattaattctccattttcttctgctatcaaaataaca gactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaa cagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcc cttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgc aggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaa catttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaataga gataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactct gaatttttttatcaagcttatcgataccgtcgacctcgagggggggcccggtacccagcttttgttccctttagtgagggttaattgcg cgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataa agtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgt cgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgac tcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggata acgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccatagg ctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggc gtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagc cactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactaga aggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaat taaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctca gcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcccca gtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgc agaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagttt gcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatca aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcag aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaa cccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgca aaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctc atgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac

The two PCR products were subjected to fusion PCR as known in the art to create the 1.5 kb fusion. The resulting fusion product was then cloned into PCR2.1TOPO to produce pCP609 (See, FIG. 16) and sequence below).

(SEQ ID NO: 143) caggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaa ggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcact atagggcgaattggagctccaccgcggtggcggccgctctagaactagtggatcccccgggctgcaggaattctccattttcttct gctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattc ccgatattggttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataatt ttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctca cggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgaca tttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatga tatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtctttt aagtaagtctactctgaatttttttaaaaggagagggtaaagagtgagaagcaaaaaattgtggatctgaaattgatacactaatgctt ttatatagggaaaaggtggtgaactactatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcga agacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcga ggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacct gccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcg caccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacc cggctcccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcg agcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgat cagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcgg agcctgctgtaaaaggatccgaatgcctctcacaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcat ctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcg ttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtg accgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagc tctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgt agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaac aacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaa aatttaacgcgaattttaacaaaattcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacg gtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgca gtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggta aggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaaga gacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggct atgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaag accgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgc agctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatccca ccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgacc accaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc aggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcga tgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatca ggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttcc gtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatc agttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaa tgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctg ccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatg gaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatata tactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtg agttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca gagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcg ctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac aggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgtt ggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagct cactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaa acagctatgaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgccctt

The plasmid PCP609 was digested with BamH1/XmaI to release the fragment containing the pAprE-aprE-stop-pSpoVG-phd construct and ligated into pBSFNASally digested with XmaI/Bcl1 to give the plasmid pCP649. FIG. 17 provides a map of pCP649. The complete sequence of pCP649 is provided below.

(SEQ ID NO: 144) tagaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttc aaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctga tgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatac ttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacag gaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttct gtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtc tactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaag agtgagaagcaaaaaattgtggatctgaaattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcga attctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgc gctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacat cgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtga tcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtca cgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggtctcgccgccaccgctggc gcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgc tcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggc caacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacggaatgcctctcacaaggatccaagcc gaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagtcgatttttacaagaattagctttatataatttctgttt ttctaaagttttatcagctacaaaagacagaaatgtattgcaatcttcaactaaatccatttgattctctccaatatgacgtttaataaattt ctgaaatacttgatttctttgttttttctcagtatacttttccatgttataacacataaaaacaacttagttttcacaaactatgacaataaaa aaagttgctttttcccctttctatgtatgttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaa aaccagtcataccaataacttaagggtaactagcctcgccggcaatagttacccttattatcaagataagaaagaaaaggatttttcg ctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatgtggtctttattcttcaactaaagcacccattagttcaacaaa cgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaag cagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattgga agagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaaca taaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttaca atagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttgga ctcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaac acctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttc tacccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggtt atcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtac tttttacagtcggttttctaatgtcactaacctgccccgttagttgaagaaggtttttatattacagctccagatccatatccttctttttctg aaccgacttctcctttttcgcttctttattccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggt cggcttaatagctcacgctatgccgacattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcggcataaatg cgtggtctaatttttatttttaataaccttgatagcaaaaaatgccattccaatacaaaaccacatacctataatcgacctgcaggaatt aattcctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcgga tgcctgtctataaaattcccgatattggcttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttattt cttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcac gataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcattta acctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctac ggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaata aattcacagaatagtcttttaagtaagtctactctgaatttttttatcaagcttatcgataccgtcgacctcgagggggggcccggtac ccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcac aattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgc gctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgc gtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg gcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctta ccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaaccc ggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagtt cttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaag agttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaa aggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaga ttatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgaca gttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa ctacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttg gtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcg gtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccat ccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggc gtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctg ggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttt tcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccg cgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcatttttta accaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaag agtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatca ccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggg gaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggt cacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactg ttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggt aacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgga gctccaccgcggtggcggccgctc

All constructs were confirmed by sequence analysis. PCR reactions were done using Hercules polymerase (Roche) as per the manufacturer's directions.

pCP649 was transformed into B. subtilis comK pnbA and integrants selected on L agar containing chloramphenicol (5 μg/ml). The activity of the expressed perhydrolase was determined by the pNB activity assay as described herein. The results indicated that the perhydrolase was expressed and active

Example 7 Expression of the Perhydrolase in Streptomyces

In this Example, experiments conducted to assess the expression of the perhydrolase in Streptomyces are described. To test expression of the perhydrolase in Streptomyces, a replicating plasmid was constructed with the phd gene being expressed from either the glucose isomerase (GIT) or the A4 promoter (See e.g., US/PCT ______, filed Nov. 18, 2004, herein incorporated by reference). However, it is not intended that the present invention be limited to these specific promoters, as any suitable promoter will find use with the present invention. Also, although the strain used for perhydrolase expression in this Example was Streptomyces lividans TK-23, it is contemplated that any Streptomyces will find use in the present invention.

The Streptomyces strains were transformed and manipulated using methods known in the art (See e.g., Kieser et al., Practical Streptomyces Genetics, John Innes [2000]).

Construction of pSECGT-MSAT and pSECA4-MSAT

Using standard methods known in the art, the phd coding sequence (See, Example 4) was cloned into pSECGT to place the gene under control of the GI promoter. Similarly, the gene was cloned in the same plasmid with the A4 promoter using methods known in the art (See e.g., USPCT ______, filed Nov. 18, 2004, herein incorporated by reference). Transformants were first selected in E. coli, verified by sequence analysis, and then transformed into S. lividans TK-23 using methods known in the art (See e.g., Kieser et al., [2000], supra). The correct clones expressed from the GI promoter and the A4 promoter were designated “pSECGT-MSAT” and “pSECA4-phd.” The sequence of pSECGT-MSAT is provided below, while FIG. 18 provides a map of the plasmid.

(SEQ ID NO: 145) ctagagtcgaccacgcaggccgccaggtagtcgacgttgatctcgcagccgagcccggccggaccggcggcgctgagcgcg aggccgacggcgggacggccggcaccggtacgcggtggcgggtcgagttcggtgagcagcccaccggcgatcaggtcgtcg acgagcgcggagacggtggcccgggtgagcccggtgacggcggcaactcccgcgcgggagagccgatctgtgctgtttgcc acggtatgcagcaccagcgcgagattatgggctcgcacgctcgactgtcggacgggggcactggaacgagaagtcaggcgag ccgtcacgcccttgacaatgccacatcctgagcaaataattcaaccactaaacaaatcaaccgcgtttcccggaggtaaccatggc caagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccc cgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcac caccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcg acctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcgg tgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgcca ccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgta cagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttc accgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacgggatccgcgagcggatc ggctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggac gtggcagtatcagcgccatgtccggcatatcccagccctccgcatgccccgaattcggcgtaatcatggtcatagctgtttcctgtg tgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagcta actcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg gtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcca gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcg acgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatc tcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaac tatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatg taggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagc cagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc agattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgtt aagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatata tgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctg actccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctca ccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcc agtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtg gtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaa aagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataa ttctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgacc gagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttc ggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttg aatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaat aaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctcttgacacatgcagctcccggagacg gtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctg gcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaa taccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagc tggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggcca gtaagcttgcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgac accaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaac cgagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaac ggaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggc ccacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgag cgtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacg taacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagc gcgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgc gatatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgac gatcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgct gccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttgg atcgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgt cctaccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaat caccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcata tgaagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgcc ctgagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgc gccatggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgc cggcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggac cgtgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccg gaccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccg gcggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtg ccaggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagcc gggcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctg ccactgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaag cgggcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcac ccctccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtc gcggaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccg cgtgcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggt cctgggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatc accgccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggacc ggctcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcga ctgatcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagacc ggatcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgat cgtcctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgct cgacgagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccg gcacggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgg gaaggcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcgg acggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctg gcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacgg cggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccg tgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggc aacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctc acggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccg acggcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctc gcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacg cggcgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgca ggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccaggga cgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccg ccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcg ccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttcc gcccgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggca gcgtcggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacg gtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggc cgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgccc gccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgc ccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcgg cagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggc gctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagc acaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccg gtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgg gccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct

FIG. 19 provides a map of pSEGT-phdA4, while the sequence is provided below:

(SEQ ID NO: 146) ctagagatcgaacttcatgttcgagttcttgttcacgtagaagccggagatgtgagaggtgatctggaactgctcaccctcgttggt ggtgacctggaggtaaagcaagtgacccttctggcggaggtggtaaggaacggggttccacggggagagagagatggccttg acggtcttgggaaggggagcttcngcgcgggggaggatggtcttgagagagggggagctagtaatgtcgtacttggacaggga gtgctccttctccgacgcatcagccacctcagcggagatggcatcgtgcagagacagacccccggaggtaaccatggccaagc gaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgt gcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaa catcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctgg tgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgt cacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctg gcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgc gctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgag gccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacaatggggatccgcgagcggatcgg ctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggacgtg gcagtatcagcgccatgtccggcatatcccagccctccgcatgccccgaattcggcgtaatcatggtcatagctgtttcctgtgtga aattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaact cacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggta tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgtt ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctca gttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcc agttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgtta agggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatat gagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctga ctccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcac cggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcca gtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtgg tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaa agcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccg agttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcg gggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttta ctttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga atactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaata aacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaa ataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctcttgacacatgcagctcccggagacggt cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctgg cttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccag taagcttgcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgaca ccaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaacc gagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaacg gaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggcc cacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgagc gtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacgt aacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagcg cgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgcg atatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgacg atcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgctg ccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttggat cgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgtcc taccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaatca ccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcatatg aagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgccct gagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgcgc catggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgccg gcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggaccg tgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccgga ccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccggc ggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtgcc aggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagccgg gcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctgcca ctgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaagcgg gcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcacccc tccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtcgcg gaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccgcgt gcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggtcct gggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatcacc gccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggaccggc tcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcgactg atcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagaccgga tcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgatcgtc ctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgctcgac gagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccggcac ggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgggaag gcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcggacgg atcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctggcac gagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacggcggc gacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccgtgac cgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggcaac gaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctcacg gcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccgacg gcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctcgca agtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacgcgg cgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgcaggc ggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccagggacgc cgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccgccg ctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcgccg agatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttccgcc cgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggcagcgt cggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacggtgg gtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggccgca ctgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgcccgccct cgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgcccac cacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcggcag caggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggcgctc cgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagcacaa cggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccggtcg gcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgggccg ccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct

Two colonies of S. lividans TK-23 pSECA4-phd were inoculated in 10 ml of TS medium+50 ppm thiostrepton and incubated at 37° C. with shaking at 200 rpm for 2 days. Three mls of broth were used to inoculate 50 ml of Streptomyces Production medium 1 and the culture was incubated for 4 days at 37° C. with shaking at 200 rpm.

A sample was taken to assay perhydrolase activity measurement as follows: 10 pls of 20 mgml lysozyme were added to 200 μl of sample. After 1 hour of incubation at 37° C., samples were centrifuged and activity was measured using the pNB activity assay described herein. SDS-PAGE and Western blots were also prepared using both clones (pSECA4-phd and pSECGT-MSAT), as known in the art. Briefly, after SDS-PAGE, the proteins were transferred to PVDF membrane and Western blot analysis was conducted. The perhydrolase was detected using an anti-perhydrolase polyclonal anti-sera (1:500 dilution) prepared against purified perhydrolase protein by Covance. The blot was developed using the ECL kit from Amersham. The results indicated that Streptomyces lividans strains were capable of expressing active perhydrolase.

Example 8 Site-Scanning Mutagenesis of the M. smegmatis Perhydrolase Gene

In this Example, experiments involving site-scanning mutagenesis of the M. smegmatis perhydrolase gene are described. In these experiments, the QuikChange® site-directed mutagenesis (QC; Stratagene) kit or the QuikChange® Multi Site-Directed mutagenesis (QCMS; Stratagene) kit was used to create site-saturation libraries at each codon in the entire M. smegmatis perhydrolase gene contained in the pMSAT-NcoI plasmid. Each perhydrolase codon was mutagenized by replacement with the NNGC (NNS; 32 combinations) degenerate codon, which encodes for all 20 amino acids and one stop codon. In the case of the QC method, complementary overlapping primers were designed for each codon of interest with 18 bases flanking the NNS codon (See, Tables 8-1 and 8-2). A comparison of cartridge purified versus unpurified primers (desalted only) revealed a better representation of amino acids in the libraries made with purified primers (15-19 amino acids versus 11-16 with unpurified primers). Thus, a majority of the libraries were created with the QC method and purified primers. A small number of the libraries were made using the QCMS method and a single 5′ phosphorylated forward primer containing 18 bases flanking both sides of the NNS codon (See, Table 8-1), however this method resulted in a greater wild type background and fewer amino acid substitutions per site compared to the QC methods. Libraries “nsa301” and “nsa302” were made using the QCMS method, but a trinucleotide mix made up of a single codon for each of the 20 amino acids (i.e., rather than 32 possibilities encoded by NNS for the 20 amino acids) was incorporated within the primers at the sites of interest.

TABLE 8-1 Site-Saturation Forward Primers Residue Primer Primer Sequence M1 nsa202F taacaggaggaattaaccnnsgccaagcgaattctgtgt (SEQ ID NO: 147) A2 nsa203F caggaggaattaaccatgnnsaagcgaattctgtgtttc (SEQ ID NO: 148) K3 nsa204F gaggaattaaccatggccnnscgaattctgtgtttcggt (SEQ ID NO: 149) R4 nsa205F gaattaaccatggccaagnnsattctgtgtttcggtgat (SEQ ID NO: 150) I5 nsa206F ttaaccatggccaagcgannsctgtgtttcggtgattcc (SEQ ID NO: 151) L6 nsa207F accatggccaagcgaattnnstgtttcggtgattccctg (SEQ ID NO: 152) C7 nsa208F atggccaagcgaattctgnnsttcggtgattccctgacc (SEQ ID NO: 153) F8 nsa209F gccaagcgaattctgtgtnnsggtgattccctgacctgg (SEQ ID NO: 154) G9 nsa210F aagcgaattctgtgtttcnnsgattccctgacctggggc (SEQ ID NO: 155) D10 nsa168F cgaattctgtgtttcggtnnstccctgacctggggctgg (SEQ ID NO: 156) S11 nsa212F attctgtgtttcggtgatnnsctgacctggggctgggtc (SEQ ID NO: 157) L12 nsa169F ctgtgtttcggtgattccnnsacctggggctgggtcccc (SEQ ID NO: 158) T13 nsa170F tgtttcggtgattccctgnnstggggctgggtccccgtc (SEQ ID NO: 159) W14 nsa171F ttcggtgattccctgaccnnsggctgggtccccgtcgaa (SEQ ID NO: 160) G15 nsa216F ggtgattccctgacctggnnstgggtccccgtcgaagac (SEQ ID NO: 161) W16 nsa172F gattccctgacctggggcnnsgtccccgtcgaagacggg (SEQ ID NO: 162) V17 nsa218F tccctgacctggggctggnnscccgtcgaagacggggca (SEQ ID NO: 163) P18 nsa219F ctgacctggggctgggtcnnsgtcgaagacggggcaccc (SEQ ID NO: 164) V19 nsa220F acctggggctgggtccccnnsgaagacggggcacccacc (SEQ ID NO: 165) E20 nsa221F tggggctgggtccccgtcnnsgacggggcacccaccgag (SEQ ID NO: 166) D21 nsa222F ggctgggtccccgtcgaannsggggcacccaccgagcgg (SEQ ID NO: 167) G22 nsa223F tgggtccccgtcgaagacnnsgcacccaccgagcggttc (SEQ ID NO: 168) A23 nsa224F gtccccgtcgaagacgggnnscccaccgagcggttcgcc (SEQ ID NO: 169) P24 nsa191F cccgtcgaagacggggcannsaccgagcggttcgccccc (SEQ ID NO: 170) T25 nsa192F gtcgaagacggggcacccnnsgagcggttcgcccccgac (SEQ ID NO: 171) E26 nsa227F gaagacggggcacccaccnnscggttcgcccccgacgtg (SEQ ID NO: 172) R27 nsa228F gacggggcacccaccgagnnsttcgcccccgacgtgcgc (SEQ ID NO: 173) F28 nsa229F ggggcacccaccgagcggnnsgcccccgacgtgcgctgg (SEQ ID NO: 174) A29 nsa230F gcacccaccgagcggttcnnscccgacgtgcgctggacc (SEQ ID NO: 175) P30 nsa231F cccaccgagcggttcgccnnsgacgtgcgctggaccggt (SEQ ID NO: 176) D31 nsa232F accgagcggttcgcccccnnsgtgcgctggaccggtgtg (SEQ ID NO: 177) V32 nsa233F gagcggttcgcccccgacnnscgctggaccggtgtgctg (SEQ ID NO: 178) R33 nsa234F cggttcgcccccgacgtgnnstggaccggtgtgctggcc (SEQ ID NO: 179) W34 nsa235F ttcgcccccgacgtgcgcnnsaccggtgtgctggcccag (SEQ ID NO: 180) T35 nsa236F gcccccgacgtgcgctggnnsggtgtgctggcccagcag (SEQ ID NO: 181) G36 nsa237F cccgacgtgcgctggaccnnsgtgctggcccagcagctc (SEQ ID NO: 182) V37 nsa238F gacgtgcgctggaccggtnnsctggcccagcagctcgga (SEQ ID NO: 183) L38 nsa239F gtgcgctggaccggtgtgnnsgcccagcagctcggagcg (SEQ ID NO: 184) A39 nsa240F cgctggaccggtgtgctgnnscagcagctcggagcggac (SEQ ID NO: 185) Q40 nsa241F tggaccggtgtgctggccnnscagctcggagcggacttc (SEQ ID NO: 186) Q41 nsa242F accggtgtgctggcccagnnsctcggagcggacttcgag (SEQ ID NO: 187) L42 nsa243F ggtgtgctggcccagcagnnsggagcggacttcgaggtg (SEQ ID NO: 188) G43 nsa244F gtgctggcccagcagctcnnsgcggacttcgaggtgatc (SEQ ID NO: 189) A44 nsa245F ctggcccagcagctcggannsgacttcgaggtgatcgag (SEQ ID NO: 190) D45 nsa246F gcccagcagctcggagcgnnsttcgaggtgatcgaggag (SEQ ID NO: 191) F46 nsa247F cagcagctcggagcggacnnsgaggtgatcgaggaggga (SEQ ID NO: 192) E47 nsa248F cagctcggagcggacttcnnsgtgatcgaggagggactg (SEQ ID NO: 193) V48 nsa249F ctcggagcggacttcgagnnsatcgaggagggactgagc (SEQ ID NO: 194) I49 nsa250F ggagcggacttcgaggtgnnsgaggagggactgagcgcg (SEQ ID NO: 195) E50 nsa251F gcggacttcgaggtgatcnnsgagggactgagcgcgcgc (SEQ ID NO: 196) E51 nsa252F gacttcgaggtgatcgagnnsggactgagcgcgcgcacc (SEQ ID NO: 197) G52 nsa253F ttcgaggtgatcgaggagnnsctgagcgcgcgcaccacc (SEQ ID NO: 198) L53 nsa193F gaggtgatcgaggagggannsagcgcgcgcaccaccaac (SEQ ID NO: 199) S54 nsa173F gtgatcgaggagggactgnnsgcgcgcaccaccaacatc (SEQ ID NO: 200) A55 nsa174F atcgaggagggactgagcnnscgcaccaccaacatcgac (SEQ ID NO: 201) R56 nsa257F gaggagggactgagcgcgnnsaccaccaacatcgacgac (SEQ ID NO: 202) T57 nsa258F gagggactgagcgcgcgcnnsaccaacatcgacgacccc (SEQ ID NO: 203) T58 nsa259F ggactgagcgcgcgcaccnnsaacatcgacgaccccacc (SEQ ID NO: 204) N59 nsa260F ctgagcgcgcgcaccaccnnsatcgacgaccccaccgat (SEQ ID NO: 205) I60 nsa261F agcgcgcgcaccaccaacnnsgacgaccccaccgatccg (SEQ ID NO: 206) D61 nsa262F gcgcgcaccaccaacatcnnsgaccccaccgatccgcgg (SEQ ID NO: 207) D62 nsa263F cgcaccaccaacatcgacnnscccaccgatccgcggctc (SEQ ID NO: 208) P63 nsa264F accaccaacatcgacgacnnsaccgatccgcggctcaac (SEQ ID NO: 209) T64 nsa194F accaacatcgacgaccccnnsgatccgcggctcaacggc (SEQ ID NO: 210) D65 nsa195F aacatcgacgaccccaccnnsccgcggctcaacggcgcg (SEQ ID NO: 211) P66 nsa267F atcgacgaccccaccgatnnscggctcaacggcgcgagc (SEQ ID NO: 212) R67 nsa196F gacgaccccaccgatccgnnsctcaacggcgcgagctac (SEQ ID NO: 213) L68 nsa269F gaccccaccgatccgcggnnsaacggcgcgagctacctg (SEQ ID NO: 214) N69 nsa270F cccaccgatccgcggctcnnsggcgcgagctacctgccg (SEQ ID NO: 215) G70 nsa271F accgatccgcggctcaacnnsgcgagctacctgccgtcg (SEQ ID NO: 216) A71 nsa272F gatccgcggctcaacggcnnsagctacctgccgtcgtgc (SEQ ID NO: 217) S72 nsa273F ccgcggctcaacggcgcgnnstacctgccgtcgtgcctc (SEQ ID NO: 218) Y73 nsa274F cggctcaacggcgcgagcnnsctgccgtcgtgcctcgcg (SEQ ID NO: 219) L74 nsa275F ctcaacggcgcgagctacnnsccgtcgtgcctcgcgacg (SEQ ID NO: 220) P75 nsa276F aacggcgcgagctacctgnnstcgtgcctcgcgacgcac (SEQ ID NO: 221) S76 nsa277F ggcgcgagctacctgccgnnstgcctcgcgacgcacctg (SEQ ID NO: 222) C77 nsa278F gcgagctacctgccgtcgnnsctcgcgacgcacctgccg (SEQ ID NO: 223) L78 nsa279F agctacctgccgtcgtgcnnsgcgacgcacctgccgctc (SEQ ID NO: 224) A79 nsa280F tacctgccgtcgtgcctcnnsacgcacctgccgctcgac (SEQ ID NO: 225) T80 nsa281F ctgccgtcgtgcctcgcgnnscacctgccgctcgacctg (SEQ ID NO: 226) H81 nsa282F ccgtcgtgcctcgcgacgnnsctgccgctcgacctggtg (SEQ ID NO: 227) L82 nsa283F tcgtgcctcgcgacgcacnnsccgctcgacctggtgatc (SEQ ID NO: 228) P83 nsa284F tgcctcgcgacgcacctgnnsctcgacctggtgatcatc (SEQ ID NO: 229) L84 nsa285F ctcgcgacgcacctgccgnnsgacctggtgatcatcatg (SEQ ID NO: 230) D85 nsa286F gcgacgcacctgccgctcnnsctggtgatcatcatgctg (SEQ ID NO: 231) L86 nsa287F acgcacctgccgctcgacnnsgtgatcatcatgctgggc (SEQ ID NO: 232) V87 nsa288F cacctgccgctcgacctgnnsatcatcatgctgggcacc (SEQ ID NO: 233) I88 nsa289F ctgccgctcgacctggtgnnsatcatgctgggcaccaac (SEQ ID NO: 234) I89 nsa290F ccgctcgacctggtgatcnnsatgctgggcaccaacgac (SEQ ID NO: 235) M90 nsa291F ctcgacctggtgatcatcnnsctgggcaccaacgacacc (SEQ ID NO: 236) L91 nsa292F gacctggtgatcatcatgnnsggcaccaacgacaccaag (SEQ ID NO: 237) G92 nsa293F ctggtgatcatcatgctgnnsaccaacgacaccaaggcc (SEQ ID NO: 238) T93 nsa294F gtgatcatcatgctgggcnnsaacgacaccaaggcctac (SEQ ID NO: 239) N94 nsa175F atcatcatgctgggcaccnnsgacaccaaggcctacttc (SEQ ID NO: 240) D95 nsa197F atcatgctgggcaccaacnnsaccaaggcctacttccgg (SEQ ID NO: 241) T96 nsa297F atgctgggcaccaacgacnnsaaggcctacttccggcgc (SEQ ID NO: 242) K97 nsa176F ctgggcaccaacgacaccnnsgcctacttccggcgcacc (SEQ ID NO: 243) A98 nsa299F ggcaccaacgacaccaagnnstacttccggcgcaccccg (SEQ ID NO: 244) Y99 nsa177F accaacgacaccaaggccnnsttccggcgcaccccgctc (SEQ ID NO: 245) F100 nsa301F aacgacaccaaggcctacXXXcggcgcaccccgctcgac (SEQ ID NO: 246) R101 nsa302F gacaccaaggcctacttcXXXcgcaccccgctcgacatc (SEQ ID NO: 247) R102 nsa303F accaaggcctacttccggnnsaccccgctcgacatcgcg (SEQ ID NO: 248) T103 nsa304F aaggcctacttccggcgcnnsccgctcgacatcgcgctg (SEQ ID NO: 249) P104 nsa305F gcctacttccggcgcaccnnsctcgacatcgcgctgggc (SEQ ID NO: 250) L105 nsa306F tacttccggcgcaccccgnnsgacatcgcgctgggcatg (SEQ ID NO: 251) D106 nsa307F ttccggcgcaccccgctcnnsatcgcgctgggcatgtcg (SEQ ID NO: 252) I107 nsa308F cggcgcaccccgctcgacnnsgcgctgggcatgtcggtg (SEQ ID NO: 253) A108 nsa309F cgcaccccgctcgacatcnnsctgggcatgtcggtgctc (SEQ ID NO: 254) L109 nsa310F accccgctcgacatcgcgnnsggcatgtcggtgctcgtc (SEQ ID NO: 255) G110 nsa311F ccgctcgacatcgcgctgnnsatgtcggtgctcgtcacg (SEQ ID NO: 256) M111 nsa312F ctcgacatcgcgctgggcnnstcggtgctcgtcacgcag (SEQ ID NO: 257) S112 nsa313F gacatcgcgctgggcatgnnsgtgctcgtcacgcaggtg (SEQ ID NO: 258) V113 nsa314F atcgcgctgggcatgtcgnnsctcgtcacgcaggtgctc (SEQ ID NO: 259) L114 nsa315F gcgctgggcatgtcggtgnnsgtcacgcaggtgctcacc (SEQ ID NO: 260) V115 nsa316F ctgggcatgtcggtgctcnnsacgcaggtgctcaccagc (SEQ ID NO: 261) T116 nsa317F ggcatgtcggtgctcgtcnnscaggtgctcaccagcgcg (SEQ ID NO: 262) Q117 nsa318F atgtcggtgctcgtcacgnnsgtgctcaccagcgcgggc (SEQ ID NO: 263) V118 nsa319F tcggtgctcgtcacgcagnnsctcaccagcgcgggcggc (SEQ ID NO: 264) L119 nsa320F gtgctcgtcacgcaggtgnnsaccagcgcgggcggcgtc (SEQ ID NO: 265) T120 nsa321F ctcgtcacgcaggtgctcnnsagcgcgggcggcgtcggc (SEQ ID NO: 266) S121 nsa322F gtcacgcaggtgctcaccnnsgcgggcggcgtcggcacc (SEQ ID NO: 267) A122 nsa323F acgcaggtgctcaccagcnnsggcggcgtcggcaccacg (SEQ ID NO: 268) G123 nsa324F caggtgctcaccagcgcgnnsggcgtcggcaccacgtac (SEQ ID NO: 269) G124 nsa325F gtgctcaccagcgcgggcnnsgtcggcaccacgtacccg (SEQ ID NO: 270) V125 nsa198F ctcaccagcgcgggcggcnnsggcaccacgtacccggca (SEQ ID NO: 271) G126 nsa327F accagcgcgggcggcgtcnnsaccacgtacccggcaccc (SEQ ID NO: 272) T127 nsa328F agcgcgggcggcgtcggcnnsacgtacccggcacccaag (SEQ ID NO: 273) T128 nsa329F gcgggcggcgtcggcaccnnstacccggcacccaaggtg (SEQ ID NO: 274) Y129 nsa330F ggcggcgtcggcaccacgnnsccggcacccaaggtgctg (SEQ ID NO: 275) P130 nsa331F ggcgtcggcaccacgtacnnsgcacccaaggtgctggtg (SEQ ID NO: 276) A131 nsa332F gtcggcaccacgtacccgnnscccaaggtgctggtggtc (SEQ ID NO: 277) P132 nsa333F ggcaccacgtacccggcannsaaggtgctggtggtctcg (SEQ ID NO: 278) K133 nsa334F accacgtacccggcacccnnsgtgctggtggtctcgccg (SEQ ID NO: 279) V134 nsa335F acgtacccggcacccaagnnsctggtggtctcgccgcca (SEQ ID NO: 280) L135 nsa336F tacccggcacccaaggtgnnsgtggtctcgccgccaccg (SEQ ID NO: 281) V136 nsa337F ccggcacccaaggtgctgnnsgtctcgccgccaccgctg (SEQ ID NO: 282) V137 nsa338F gcacccaaggtgctggtgnnstcgccgccaccgctggcg (SEQ ID NO: 283) S138 nsa339F cccaaggtgctggtggtcnnsccgccaccgctggcgccc (SEQ ID NO: 284) P139 nsa340F aaggtgctggtggtctcgnnsccaccgctggcgcccatg (SEQ ID NO: 285) P140 nsa341F gtgctggtggtctcgccgnnsccgctggcgcccatgccg (SEQ ID NO: 286) P141 nsa342F ctggtggtctcgccgccannsctggcgcccatgccgcac (SEQ ID NO: 287) L142 nsa343F gtggtctcgccgccaccgnnsgcgcccatgccgcacccc (SEQ ID NO: 288) A143 nsa344F gtctcgccgccaccgctgnnscccatgccgcacccctgg (SEQ ID NO: 289) P144 nsa345F tcgccgccaccgctggcgnnsatgccgcacccctggttc (SEQ ID NO: 290) M145 nsa346F ccgccaccgctggcgcccnnsccgcacccctggttccag (SEQ ID NO: 291) P146 nsa178F ccaccgctggcgcccatgnnscacccctggttccagttg (SEQ ID NO: 292) H147 nsa348F ccgctggcgcccatgccgnnsccctggttccagttgatc (SEQ ID NO: 293) P148 nsa199F ctggcgcccatgccgcacnnstggttccagttgatcttc (SEQ ID NO: 294) W149 nsa179F gcgcccatgccgcaccccnnsttccagttgatcttcgag (SEQ ID NO: 295) F150 nsa180F cccatgccgcacccctggnnscagttgatcttcgagggc (SEQ ID NO: 296) Q151 nsa352F atgccgcacccctggttcnnsttgatcttcgagggcggc (SEQ ID NO: 297) L152 nsa353F ccgcacccctggttccagnnsatcttcgagggcggcgag (SEQ ID NO: 298) I153 nsa200F cacccctggttccagttgnnsttcgagggcggcgagcag (SEQ ID NO: 299) F154 nsa201F ccctggttccagttgatcnnsgagggcggcgagcagaag (SEQ ID NO: 300) E155 nsa356F tggttccagttgatcttcnnsggcggcgagcagaagacc (SEQ ID NO: 301) G156 nsa357F ttccagttgatcttcgagnnsggcgagcagaagaccact (SEQ ID NO: 302) G157 nsa358F cagttgatcttcgagggcnnsgagcagaagaccactgag (SEQ ID NO: 303) E158 nsa359F ttgatcttcgagggcggcnnscagaagaccactgagctc (SEQ ID NO: 304) Q159 nsa360F atcttcgagggcggcgagnnsaagaccactgagctcgcc (SEQ ID NO: 305) K160 nsa361F ttcgagggcggcgagcagnnsaccactgagctcgcccgc (SEQ ID NO: 306) T161 nsa362F gagggcggcgagcagaagnnsactgagctcgcccgcgtg (SEQ ID NO: 307) T162 nsa363F ggcggcgagcagaagaccnnsgagctcgcccgcgtgtac (SEQ ID NO: 308) E163 nsa364F ggcgagcagaagaccactnnsctcgcccgcgtgtacagc (SEQ ID NO: 309) L164 nsa365F gagcagaagaccactgagnnsgcccgcgtgtacagcgcg (SEQ ID NO: 310) A165 nsa366F cagaagaccactgagctcnnscgcgtgtacagcgcgctc (SEQ ID NO: 311) R166 nsa367F aagaccactgagctcgccnnsgtgtacagcgcgctcgcg (SEQ ID NO: 312) V167 nsa368F accactgagctcgcccgcnnstacagcgcgctcgcgtcg (SEQ ID NO: 313) Y168 nsa369F actgagctcgcccgcgtgnnsagcgcgctcgcgtcgttc (SEQ ID NO: 314) S169 nsa370F gagctcgcccgcgtgtacnnsgcgctcgcgtcgttcatg (SEQ ID NO: 315) A170 nsa371F ctcgcccgcgtgtacagcnnsctcgcgtcgttcatgaag (SEQ ID NO: 316) L171 nsa372F gcccgcgtgtacagcgcgnnsgcgtcgttcatgaaggtg (SEQ ID NO: 317) A172 nsa373F cgcgtgtacagcgcgctcnnstcgttcatgaaggtgccg (SEQ ID NO: 318) S173 nsa374F gtgtacagcgcgctcgcgnnsttcatgaaggtgccgttc (SEQ ID NO: 319) F174 nsa375F tacagcgcgctcgcgtcgnnsatgaaggtgccgttcttc (SEQ ID NO: 320) M175 nsa376F agcgcgctcgcgtcgttcnnsaaggtgccgttcttcgac (SEQ ID NO: 321) K176 nsa377F gcgctcgcgtcgttcatgnnsgtgccgttcttcgacgcg (SEQ ID NO: 322) V177 nsa378F ctcgcgtcgttcatgaagnnsccgttcttcgacgcgggt (SEQ ID NO: 323) P178 nsa379F gcgtcgttcatgaaggtgnnsttcttcgacgcgggttcg (SEQ ID NO: 324) F179 nsa380F tcgttcatgaaggtgccgnnsttcgacgcgggttcggtg (SEQ ID NO: 325) F180 nsa381F ttcatgaaggtgccgttcnnsgacgcgggttcggtgatc (SEQ ID NO: 326) D181 nsa382F atgaaggtgccgttcttcnnsgcgggttcggtgatcagc (SEQ ID NO: 327) A182 nsa383F aaggtgccgttcttcgacnnsggttcggtgatcagcacc (SEQ ID NO: 328) G183 nsa384F gtgccgttcttcgacgcgnnstcggtgatcagcaccgac (SEQ ID NO: 329) S184 nsa385F ccgttcttcgacgcgggtnnsgtgatcagcaccgacggc (SEQ ID NO: 330) V185 nsa386F ttcttcgacgcgggttcgnnsatcagcaccgacggcgtc (SEQ ID NO: 331) I186 nsa387F ttcgacgcgggttcggtgnnsagcaccgacggcgtcgac (SEQ ID NO: 332) S187 nsa388F gacgcgggttcggtgatcnnsaccgacggcgtcgacgga (SEQ ID NO: 333) T188 nsa389F gcgggttcggtgatcagcnnsgacggcgtcgacggaatc (SEQ ID NO: 334) D189 nsa390F ggttcggtgatcagcaccnnsggcgtcgacggaatccac (SEQ ID NO: 335) G190 nsa391F tcggtgatcagcaccgacnnsgtcgacggaatccacttc (SEQ ID NO: 336) V191 nsa392F gtgatcagcaccgacggcnnsgacggaatccacttcacc (SEQ ID NO: 337) D192 nsa393F atcagcaccgacggcgtcnnsggaatccacttcaccgag (SEQ ID NO: 338) G193 nsa394F agcaccgacggcgtcgacnnsatccacttcaccgaggcc (SEQ ID NO: 339) I194 nsa181F accgacggcgtcgacggannscacttcaccgaggccaac (SEQ ID NO: 340) H195 nsa396F gacggcgtcgacggaatcnnsttcaccgaggccaacaat (SEQ ID NO: 341) F196 nsa182F ggcgtcgacggaatccacnnsaccgaggccaacaatcgc (SEQ ID NO: 342) T197 nsa398F gtcgacggaatccacttcnnsgaggccaacaatcgcgat (SEQ ID NO: 343) E198 nsa399F gacggaatccacttcaccnnsgccaacaatcgcgatctc (SEQ ID NO: 344) A199 nsa400F ggaatccacttcaccgagnnsaacaatcgcgatctcggg (SEQ ID NO: 345) N200 nsa401F atccacttcaccgaggccnnsaatcgcgatctcggggtg (SEQ ID NO: 346) N201 nsa402F cacttcaccgaggccaacnnscgcgatctcggggtggcc (SEQ ID NO: 347) R202 nsa403F ttcaccgaggccaacaatnnsgatctcggggtggccctc (SEQ ID NO: 348) D203 nsa404F accgaggccaacaatcgcnnsctcggggtggccctcgcg (SEQ ID NO: 349) L204 nsa405F gaggccaacaatcgcgatnnsggggtggccctcgcggaa (SEQ ID NO: 350) G205 nsa406F gccaacaatcgcgatctcnnsgtggccctcgcggaacag (SEQ ID NO: 351) V206 nsa407F aacaatcgcgatctcgggnnsgccctcgcggaacaggtg (SEQ ID NO: 352) A207 nsa408F aatcgcgatctcggggtgnnsctcgcggaacaggtgcag (SEQ ID NO: 353) L208 nsa409F cgcgatctcggggtggccnnsgcggaacaggtgcagagc (SEQ ID NO: 354) A209 nsa410F gatctcggggtggccctcnnsgaacaggtgcagagcctg (SEQ ID NO: 355) E210 nsa411F ctcggggtggccctcgcgnnscaggtgcagagcctgctg (SEQ ID NO: 356) Q211 nsa412F ggggtggccctcgcggaannsgtgcagagcctgctgtaa (SEQ ID NO: 357) V212 nsa413F gtggccctcgcggaacagnnscagagcctgctgtaaaag (SEQ ID NO: 358) Q213 nsa414F gccctcgcggaacaggtgnnsagcctgctgtaaaagggc (SEQ ID NO: 359) S214 nsa415F ctcgcggaacaggtgcagnnsctgctgtaaaagggcgaa (SEQ ID NO: 360) L215 nsa416F gcggaacaggtgcagagcnnsctgtaaaagggcgaattc (SEQ ID NO: 361) L216 nsa417F gaacaggtgcagagcctgnnstaaaagggcgaattctgc (SEQ ID NO: 362)

TABLE 8-2 Site-Saturation Reverse Primer Sequences Residue Primer Primer Sequence M1 nsa202R ACACAGAATTCGCTTGGCSNNGGTTAATTCCTCCTGTTA (SEQ ID NO: 363) A2 nsa203R GAAACACAGAATTCGCTTSNNCATGGTTAATTCCTCCTG (SEQ ID NO: 364) K3 nsa204R ACCGAAACACAGAATTCGSNNGGCCATGGTTAATTCCTC (SEQ ID NO: 365) R4 nsa205R ATCACCGAAACACAGAATSNNCTTGGCCATGGTTAATTC (SEQ ID NO: 366) I5 nsa206R GGAATCACCGAAACACAGSNNTCGCTTGGCCATGGTTAA (SEQ ID NO: 367) L6 nsa207R CAGGGAATCACCGAAACASNNAATTCGCTTGGCCATGGT (SEQ ID NO: 368) C7 nsa208R GGTCAGGGAATCACCGAASNNCAGAATTCGCTTGGCCAT (SEQ ID NO: 369) F8 nsa209R CCAGGTCAGGGAATCACCSNNACACAGAATTCGCTTGGC (SEQ ID NO: 370) G9 nsa210R GCCCCAGGTCAGGGAATCSNNGAAACACAGAATTCGCTT (SEQ ID NO: 371) D10 nsa168R CCAGCCCCAGGTCAGGGASNNACCGAAACACAGAATTCG (SEQ ID NO: 372) S11 nsa212R GACCCAGCCCCAGGTCAGSNNATCACCGAAACACAGAAT (SEQ ID NO: 373) L12 nsa169R GGGGACCCAGCCCCAGGTSNNGGAATCACCGAAACACAG (SEQ ID NO: 374) T13 nsa170R GACGGGGACCCAGCCCCASNNCAGGGAATCACCGAAACA (SEQ ID NO: 375) W14 nsa171R TTCGACGGGGACCCAGCCSNNGGTCAGGGAATCACCGAA (SEQ ID NO: 376) G15 nsa216R GTCTTCGACGGGGACCCASNNCCAGGTCAGGGAATCACC (SEQ ID NO: 377) W16 nsa172R CCCGTCTTCGACGGGGACSNNGCCCCAGGTCAGGGAATC (SEQ ID NO: 378) V17 nsa218R TGCCCCGTCTTCGACGGGSNNCCAGCCCCAGGTCAGGGA (SEQ ID NO: 379) P18 nsa219R GGGTGCCCCGTCTTCGACSNNGACCCAGCCCCAGGTCAG (SEQ ID NO: 380) V19 nsa220R GGTGGGTGCCCCGTCTTCSNNGGGGACCCAGCCCCAGGT (SEQ ID NO: 381) E20 nsa221R CTCGGTGGGTGCCCCGTCSNNGACGGGGACCCAGCCCCA (SEQ ID NO: 382) D21 nsa222R CCGCTCGGTGGGTGCCCCSNNTTCGACGGGGACCCAGCC (SEQ ID NO: 383) G22 nsa223R GAACCGCTCGGTGGGTGCSNNGTCTTCGACGGGGACCCA (SEQ ID NO: 384) A23 nsa224R GGCGAACCGCTCGGTGGGSNNCCCGTCTTCGACGGGGAC (SEQ ID NO: 385) P24 nsa191R GGGGGCGAACCGCTCGGTSNNTGCCCCGTCTTCGACGGG (SEQ ID NO: 386) T25 nsa192R GTCGGGGGCGAACCGCTCSNNGGGTGCCCCGTCTTCGAC (SEQ ID NO: 387) E26 nsa227R CACGTCGGGGGCGAACCGSNNGGTGGGTGCCCCGTCTTC (SEQ ID NO: 388) R27 nsa228R GCGCACGTCGGGGGCGAASNNCTCGGTGGGTGCCCCGTC (SEQ ID NO: 389) F28 nsa229R CCAGCGCACGTCGGGGGCSNNCCGCTCGGTGGGTGCCCC (SEQ ID NO: 390) A29 nsa230R GGTCCAGCGCACGTCGGGSNNGAACCGCTCGGTGGGTGC (SEQ ID NO: 391) P30 nsa231R ACCGGTCCAGCGCACGTCSNNGGCGAACCGCTCGGTGGG (SEQ ID NO: 392) D31 nsa232R CACACCGGTCCAGCGCACSNNGGGGGCGAACCGCTCGGT (SEQ ID NO: 393) V32 nsa233R CAGCACACCGGTCCAGCGSNNGTCGGGGGCGAACCGCTC (SEQ ID NO: 394) R33 nsa234R GGCCAGCACACCGGTCCASNNCACGTCGGGGGCGAACCG (SEQ ID NO: 395) W34 nsa235R CTGGGCCAGCACACCGGTSNNGCGCACGTCGGGGGCGAA (SEQ ID NO: 396) T35 nsa236R CTGCTGGGCCAGCACACCSNNCCAGCGCACGTCGGGGGC (SEQ ID NO: 397) G36 nsa237R GAGCTGCTGGGCCAGCACSNNGGTCCAGCGCACGTCGGG (SEQ ID NO: 398) V37 nsa238R TCCGAGCTGCTGGGCCAGSNNACCGGTCCAGCGCACGTC (SEQ ID NO: 399) L38 nsa239R CGCTCCGAGCTGCTGGGCSNNCACACCGGTCCAGCGCAC (SEQ ID NO: 400) A39 nsa240R GTCCGCTCCGAGCTGCTGSNNCAGCACACCGGTCCAGCG (SEQ ID NO: 401) Q40 nsa241R GAAGTCCGCTCCGAGCTGSNNGGCCAGCACACCGGTCCA (SEQ ID NO: 402) Q41 nsa242R CTCGAAGTCCGCTCCGAGSNNCTGGGCCAGCACACCGGT (SEQ ID NO: 403) L42 nsa243R CACCTCGAAGTCCGCTCCSNNCTGCTGGGCCAGCACACC (SEQ ID NO: 404) G43 nsa244R GATCACCTCGAAGTCCGCSNNGAGCTGCTGGGCCAGCAC (SEQ ID NO: 405) A44 nsa245R CTCGATCACCTCGAAGTCSNNTCCGAGCTGCTGGGCCAG (SEQ ID NO: 406) D45 nsa246R CTCCTCGATCACCTCGAASNNCGCTCCGAGCTGCTGGGC (SEQ ID NO: 407) F46 nsa247R TCCCTCCTCGATCACCTCSNNGTCCGCTCCGAGCTGCTG (SEQ ID NO: 408) E47 nsa248R CAGTCCCTCCTCGATCACSNNGAAGTCCGCTCCGAGCTG (SEQ ID NO: 409) V48 nsa249R GCTCAGTCCCTCCTCGATSNNCTCGAAGTCCGCTCCGAG (SEQ ID NO: 410) I49 nsa250R CGCGCTCAGTCCCTCCTCSNNCACCTCGAAGTCCGCTCC (SEQ ID NO: 411) E50 nsa251R GCGCGCGCTCAGTCCCTCSNNGATCACCTCGAAGTCCGC (SEQ ID NO: 412) E51 nsa252R GGTGCGCGCGCTCAGTCCSNNCTCGATCACCTCGAAGTC (SEQ ID NO: 413) G52 nsa253R GGTGGTGCGCGCGCTCAGSNNCTCCTCGATCACCTCGAA (SEQ ID NO: 414) L53 nsa193R GTTGGTGGTGCGCGCGCTSNNTCCCTCCTCGATCACCTC (SEQ ID NO: 415) S54 nsa173R GATGTTGGTGGTGCGCGCSNNCAGTCCCTCCTCGATCAC (SEQ ID NO: 416) A55 nsa174R GTCGATGTTGGTGGTGCGSNNGCTCAGTCCCTCCTCGAT (SEQ ID NO: 417) R56 nsa257R GTCGTCGATGTTGGTGGTSNNCGCGCTCAGTCCCTCCTC (SEQ ID NO: 418) T57 nsa258R GGGGTCGTCGATGTTGGTSNNGCGCGCGCTCAGTCCCTC (SEQ ID NO: 419) T58 nsa259R GGTGGGGTCGTCGATGTTSNNGGTGCGCGCGCTCAGTCC (SEQ ID NO: 420) N59 nsa260R ATCGGTGGGGTCGTCGATSNNGGTGGTGCGCGCGCTCAG (SEQ ID NO: 421) I60 nsa261R CGGATCGGTGGGGTCGTCSNNGTTGGTGGTGCGCGCGCT (SEQ ID NO: 422) D61 nsa262R CCGCGGATCGGTGGGGTCSNNGATGTTGGTGGTGCGCGC (SEQ ID NO: 423) D62 nsa263R GAGCCGCGGATCGGTGGGSNNGTCGATGTTGGTGGTGCG (SEQ ID NO: 424) P63 nsa264R GTTGAGCCGCGGATCGGTSNNGTCGTCGATGTTGGTGGT (SEQ ID NO: 425) T64 nsa194R GCCGTTGAGCCGCGGATCSNNGGGGTCGTCGATGTTGGT (SEQ ID NO: 426) D65 nsa195R CGCGCCGTTGAGCCGCGGSNNGGTGGGGTCGTCGATGTT (SEQ ID NO: 427) P66 nsa267R GCTCGCGCCGTTGAGCCGSNNATCGGTGGGGTCGTCGAT (SEQ ID NO: 428) R67 nsa196R GTAGCTCGCGCCGTTGAGSNNCGGATCGGTGGGGTCGTC (SEQ ID NO: 429) L68 nsa269R CAGGTAGCTCGCGCCGTTSNNCCGCGGATCGGTGGGGTC (SEQ ID NO: 430) N69 nsa270R CGGCAGGTAGCTCGCGCCSNNGAGCCGCGGATCGGTGGG (SEQ ID NO: 431) G70 nsa271R CGACGGCAGGTAGCTCGCSNNGTTGAGCCGCGGATCGGT (SEQ ID NO: 432) A71 nsa272R GCACGACGGCAGGTAGCTSNNGCCGTTGAGCCGCGGATC (SEQ ID NO: 433) S72 nsa273R GAGGCACGACGGCAGGTASNNCGCGCCGTTGAGCCGCGG (SEQ ID NO: 434) Y73 nsa274R CGCGAGGCACGACGGCAGSNNGCTCGCGCCGTTGAGCCG (SEQ ID NO: 435) L74 nsa275R CGTCGCGAGGCACGACGGSNNGTAGCTCGCGCCGTTGAG (SEQ ID NO: 436) P75 nsa276R GTGCGTCGCGAGGCACGASNNCAGGTAGCTCGCGCCGTT (SEQ ID NO: 437) S76 nsa277R CAGGTGCGTCGCGAGGCASNNCGGCAGGTAGCTCGCGCC (SEQ ID NO: 438) C77 nsa278R CGGCAGGTGCGTCGCGAGSNNCGACGGCAGGTAGCTCGC (SEQ ID NO: 439) L78 nsa279R GAGCGGCAGGTGCGTCGCSNNGCACGACGGCAGGTAGCT (SEQ ID NO: 440) A79 nsa280R GTCGAGCGGCAGGTGCGTSNNGAGGCACGACGGCAGGTA (SEQ ID NO: 441) T80 nsa281R CAGGTCGAGCGGCAGGTGSNNCGCGAGGCACGACGGCAG (SEQ ID NO: 442) H81 nsa282R CACCAGGTCGAGCGGCAGSNNCGTCGCGAGGCACGACGG (SEQ ID NO: 443) L82 nsa283R GATCACCAGGTCGAGCGGSNNGTGCGTCGCGAGGCACGA (SEQ ID NO: 444) P83 nsa284R GATGATCACCAGGTCGAGSNNCAGGTGCGTCGCGAGGCA (SEQ ID NO: 445) L84 nsa285R CATGATGATCACCAGGTCSNNCGGCAGGTGCGTCGCGAG (SEQ ID NO: 446) D85 nsa286R CAGCATGATGATCACCAGSNNGAGCGGCAGGTGCGTCGC (SEQ ID NO: 447) L86 nsa287R GCCCAGCATGATGATCACSNNGTCGAGCGGCAGGTGCGT (SEQ ID NO: 448) V87 nsa288R GGTGCCCAGCATGATGATSNNCAGGTCGAGCGGCAGGTG (SEQ ID NO: 449) I88 nsa289R GTTGGTGCCCAGCATGATSNNCACCAGGTCGAGCGGCAG (SEQ ID NO: 450) I89 nsa290R GTCGTTGGTGCCCAGCATSNNGATCACCAGGTCGAGCGG (SEQ ID NO: 451) M90 nsa291R GGTGTCGTTGGTGCCCAGSNNGATGATCACCAGGTCGAG (SEQ ID NO: 452) L91 nsa292R CTTGGTGTCGTTGGTGCCSNNCATGATGATCACCAGGTC (SEQ ID NO: 453) G92 nsa293R GGCCTTGGTGTCGTTGGTSNNCAGCATGATGATCACCAG (SEQ ID NO: 454) T93 nsa294R GTAGGCCTTGGTGTCGTTSNNGCCCAGCATGATGATCAC (SEQ ID NO: 455) N94 nsa175R GAAGTAGGCCTTGGTGTCSNNGGTGCCCAGCATGATGAT (SEQ ID NO: 456) D95 nsa197R CCGGAAGTAGGCCTTGGTSNNGTTGGTGCCCAGCATGAT (SEQ ID NO: 457) T96 nsa297R GCGCCGGAAGTAGGCCTTSNNGTCGTTGGTGCCCAGCAT (SEQ ID NO: 458) K97 nsa176R GGTGCGCCGGAAGTAGGCSNNGGTGTCGTTGGTGCCCAG (SEQ ID NO: 459) A98 nsa299R CGGGGTGCGCCGGAAGTASNNCTTGGTGTCGTTGGTGCC (SEQ ID NO: 460) Y99 nsa177R GAGCGGGGTGCGCCGGAASNNGGCCTTGGTGTCGTTGGT (SEQ ID NO: 461) F100 nsa301R GTCGAGCGGGGTGCGCCGSNNGTAGGCCTTGGTGTCGTT (SEQ ID NO: 462) R101 nsa302R GATGTCGAGCGGGGTGCGSNNGAAGTAGGCCTTGGTGTC (SEQ ID NO: 463) R102 nsa303R CGCGATGTCGAGCGGGGTSNNCCGGAAGTAGGCCTTGGT (SEQ ID NO: 464) T103 nsa304R CAGCGCGATGTCGAGCGGSNNGCGCCGGAAGTAGGCCTT (SEQ ID NO: 465) P104 nsa305R GCCCAGCGCGATGTCGAGSNNGGTGCGCCGGAAGTAGGC (SEQ ID NO: 466) L105 nsa306R CATGCCCAGCGCGATGTCSNNCGGGGTGCGCCGGAAGTA (SEQ ID NO: 467) D106 nsa307R CGACATGCCCAGCGCGATSNNGAGCGGGGTGCGCCGGAA (SEQ ID NO: 468) I107 nsa308R CACCGACATGCCCAGCGCSNNGTCGAGCGGGGTGCGCCG (SEQ ID NO: 469) A108 nsa309R GAGCACCGACATGCCCAGSNNGATGTCGAGCGGGGTGCG (SEQ ID NO: 470) L109 nsa310R GACGAGCACCGACATGCCSNNCGCGATGTCGAGCGGGGT (SEQ ID NO: 471) G110 nsa311R CGTGACGAGCACCGACATSNNCAGCGCGATGTCGAGCGG (SEQ ID NO: 472) M111 nsa312R CTGCGTGACGAGCACCGASNNGCCCAGCGCGATGTCGAG (SEQ ID NO: 473) S112 nsa313R CACCTGCGTGACGAGCACSNNCATGCCCAGCGCGATGTC (SEQ ID NO: 474) V113 nsa314R GAGCACCTGCGTGACGAGSNNCGACATGCCCAGCGCGAT (SEQ ID NO: 475) L114 nsa315R GGTGAGCACCTGCGTGACSNNCACCGACATGCCCAGCGC (SEQ ID NO: 476) V115 nsa316R GCTGGTGAGCACCTGCGTSNNGAGCACCGACATGCCCAG (SEQ ID NO: 477) T116 nsa317R CGCGCTGGTGAGCACCTGSNNGACGAGCACCGACATGCC (SEQ ID NO: 478) Q117 nsa318R GCCCGCGCTGGTGAGCACSNNCGTGACGAGCACCGACAT (SEQ ID NO: 479) V118 nsa319R GCCGCCCGCGCTGGTGAGSNNCTGCGTGACGAGCACCGA (SEQ ID NO: 480) L119 nsa320R GACGCCGCCCGCGCTGGTSNNCACCTGCGTGACGAGCAC (SEQ ID NO: 481) T120 nsa321R GCCGACGCCGCCCGCGCTSNNGAGCACCTGCGTGACGAG (SEQ ID NO: 482) S121 nsa322R GGTGCCGACGCCGCCCGCSNNGGTGAGCACCTGCGTGAC (SEQ ID NO: 483) A122 nsa323R CGTGGTGCCGACGCCGCCSNNGCTGGTGAGCACCTGCGT (SEQ ID NO: 484) G123 nsa324R GTACGTGGTGCCGACGCCSNNCGCGCTGGTGAGCACCTG (SEQ ID NO: 485) G124 nsa325R CGGGTACGTGGTGCCGACSNNGCCCGCGCTGGTGAGCAC (SEQ ID NO: 486) V125 nsa198R TGCCGGGTACGTGGTGCCSNNGCCGCCCGCGCTGGTGAG (SEQ ID NO: 487) G126 nsa327R GGGTGCCGGGTACGTGGTSNNGACGCCGCCCGCGCTGGT (SEQ ID NO: 488) T127 nsa328R CTTGGGTGCCGGGTACGTSNNGCCGACGCCGCCCGCGCT (SEQ ID NO: 489) T128 nsa329R CACCTTGGGTGCCGGGTASNNGGTGCCGACGCCGCCCGC (SEQ ID NO: 490) Y129 nsa330R CAGCACCTTGGGTGCCGGSNNCGTGGTGCCGACGCCGCC (SEQ ID NO: 491) P130 nsa331R CACCAGCACCTTGGGTGCSNNGTACGTGGTGCCGACGCC (SEQ ID NO: 492) A131 nsa332R GACCACCAGCACCTTGGGSNNCGGGTACGTGGTGCCGAC (SEQ ID NO: 493) P132 nsa333R CGAGACCACCAGCACCTTSNNTGCCGGGTACGTGGTGCC (SEQ ID NO: 494) K133 nsa334R CGGCGAGACCACCAGCACSNNGGGTGCCGGGTACGTGGT (SEQ ID NO: 495) V134 nsa335R TGGCGGCGAGACCACCAGSNNCTTGGGTGCCGGGTACGT (SEQ ID NO: 496) L135 nsa336R CGGTGGCGGCGAGACCACSNNCACCTTGGGTGCCGGGTA (SEQ ID NO: 497) V136 nsa337R CAGCGGTGGCGGCGAGACSNNCAGCACCTTGGGTGCCGG (SEQ ID NO: 498) V137 nsa338R CGCCAGCGGTGGCGGCGASNNCACCAGCACCTTGGGTGC (SEQ ID NO: 499) S138 nsa339R GGGCGCCAGCGGTGGCGGSNNGACCACCAGCACCTTGGG (SEQ ID NO: 500) P139 nsa340R CATGGGCGCCAGCGGTGGSNNCGAGACCACCAGCACCTT (SEQ ID NO: 501) P140 nsa341R CGGCATGGGCGCCAGCGGSNNCGGCGAGACCACCAGCAC (SEQ ID NO: 502) P141 nsa342R GTGCGGCATGGGCGCCAGSNNTGGCGGCGAGACCACCAG (SEQ ID NO: 503) L142 nsa343R GGGGTGCGGCATGGGCGCSNNCGGTGGCGGCGAGACCAC (SEQ ID NO: 504) A143 nsa344R CCAGGGGTGCGGCATGGGSNNCAGCGGTGGCGGCGAGAC (SEQ ID NO: 505) P144 nsa345R GAACCAGGGGTGCGGCATSNNCGCCAGCGGTGGCGGCGA (SEQ ID NO: 506) M145 nsa346R CTGGAACCAGGGGTGCGGSNNGGGCGCCAGCGGTGGCGG (SEQ ID NO: 507) P146 nsa178R CAACTGGAACCAGGGGTGSNNCATGGGCGCCAGCGGTGG (SEQ ID NO: 508) H147 nsa348R GATCAACTGGAACCAGGGSNNCGGCATGGGCGCCAGCGG (SEQ ID NO: 509) P148 nsa199R GAAGATCAACTGGAACCASNNGTGCGGCATGGGCGCCAG (SEQ ID NO: 510) W149 nsa179R CTCGAAGATCAACTGGAASNNGGGGTGCGGCATGGGCGC (SEQ ID NO: 511) F150 nsa180R GCCCTCGAAGATCAACTGSNNCCAGGGGTGCGGCATGGG (SEQ ID NO: 512) Q151 nsa352R GCCGCCCTCGAAGATCAASNNGAACCAGGGGTGCGGCAT (SEQ ID NO: 513) L152 nsa353R CTCGCCGCCCTCGAAGATSNNCTGGAACCAGGGGTGCGG (SEQ ID NO: 514) I153 nsa200R CTGCTCGCCGCCCTCGAASNNCAACTGGAACCAGGGGTG (SEQ ID NO: 515) F154 nsa201R CTTCTGCTCGCCGCCCTCSNNGATCAACTGGAACCAGGG (SEQ ID NO: 516) E155 nsa356R GGTCTTCTGCTCGCCGCCSNNGAAGATCAACTGGAACCA (SEQ ID NO: 517) G156 nsa357R AGTGGTCTTCTGCTCGCCSNNCTCGAAGATCAACTGGAA (SEQ ID NO: 518) G157 nsa358R CTCAGTGGTCTTCTGCTCSNNGCCCTCGAAGATCAACTG (SEQ ID NO: 519) E158 nsa359R GAGCTCAGTGGTCTTCTGSNNGCCGCCCTCGAAGATCAA (SEQ ID NO: 520) Q159 nsa360R GGCGAGCTCAGTGGTCTTSNNCTCGCCGCCCTCGAAGAT (SEQ ID NO: 521) K160 nsa361R GCGGGCGAGCTCAGTGGTSNNCTGCTCGCCGCCCTCGAA (SEQ ID NO: 522) T161 nsa362R CACGCGGGCGAGCTCAGTSNNCTTCTGCTCGCCGCCCTC (SEQ ID NO: 523) T162 nsa363R GTACACGCGGGCGAGCTCSNNGGTCTTCTGCTCGCCGCC (SEQ ID NO: 524) E163 nsa364R GCTGTACACGCGGGCGAGSNNAGTGGTCTTCTGCTCGCC (SEQ ID NO: 525) L164 nsa365R CGCGCTGTACACGCGGGCSNNCTCAGTGGTCTTCTGCTC (SEQ ID NO: 526) A165 nsa366R GAGCGCGCTGTACACGCGSNNGAGCTCAGTGGTCTTCTG (SEQ ID NO: 527) R166 nsa367R CGCGAGCGCGCTGTACACSNNGGCGAGCTCAGTGGTCTT (SEQ ID NO: 528) V167 nsa368R CGACGCGAGCGCGCTGTASNNGCGGGCGAGCTCAGTGGT (SEQ ID NO: 529) Y168 nsa369R GAACGACGCGAGCGCGCTSNNCACGCGGGCGAGCTCAGT (SEQ ID NO: 530) S169 nsa370R CATGAACGACGCGAGCGCSNNGTACACGCGGGCGAGCTC (SEQ ID NO: 531) A170 nsa371R CTTCATGAACGACGCGAGSNNGCTGTACACGCGGGCGAG (SEQ ID NO: 532) L171 nsa372R CACCTTCATGAACGACGCSNNCGCGCTGTACACGCGGGC (SEQ ID NO: 533) A172 nsa373R CGGCACCTTCATGAACGASNNGAGCGCGCTGTACACGCG (SEQ ID NO: 534) S173 nsa374R GAACGGCACCTTCATGAASNNCGCGAGCGCGCTGTACAC (SEQ ID NO: 535) F174 nsa375R GAAGAACGGCACCTTCATSNNCGACGCGAGCGCGCTGTA (SEQ ID NO: 536) M175 nsa376R GTCGAAGAACGGCACCTTSNNGAACGACGCGAGCGCGCT (SEQ ID NO: 537) K176 nsa377R CGCGTCGAAGAACGGCACSNNCATGAACGACGCGAGCGC (SEQ ID NO: 538) V177 nsa378R ACCCGCGTCGAAGAACGGSNNCTTCATGAACGACGCGAG (SEQ ID NO: 539) P178 nsa379R CGAACCCGCGTCGAAGAASNNCACCTTCATGAACGACGC (SEQ ID NO: 540) F179 nsa380R CACCGAACCCGCGTCGAASNNCGGCACCTTCATGAACGA (SEQ ID NO: 541) F180 nsa381R GATCACCGAACCCGCGTCSNNGAACGGCACCTTCATGAA (SEQ ID NO: 542) D181 nsa382R GCTGATCACCGAACCCGCSNNGAAGAACGGCACCTTCAT (SEQ ID NO: 543) A182 nsa383R GGTGCTGATCACCGAACCSNNGTCGAAGAACGGCACCTT (SEQ ID NO: 544) G183 nsa384R GTCGGTGCTGATCACCGASNNCGCGTCGAAGAACGGCAC (SEQ ID NO: 545) S184 nsa385R GCCGTCGGTGCTGATCACSNNACCCGCGTCGAAGAACGG (SEQ ID NO: 546) V185 nsa386R GACGCCGTCGGTGCTGATSNNCGAACCCGCGTCGAAGAA (SEQ ID NO: 547) I186 nsa387R GTCGACGCCGTCGGTGCTSNNCACCGAACCCGCGTCGAA (SEQ ID NO: 548) S187 nsa388R TCCGTCGACGCCGTCGGTSNNGATCACCGAACCCGCGTC (SEQ ID NO: 549) T188 nsa389R GATTCCGTCGACGCCGTCSNNGCTGATCACCGAACCCGC (SEQ ID NO: 550) D189 nsa390R GTGGATTCCGTCGACGCCSNNGGTGCTGATCACCGAACC (SEQ ID NO: 551) G190 nsa391R GAAGTGGATTCCGTCGACSNNGTCGGTGCTGATCACCGA (SEQ ID NO: 552) V191 nsa392R GGTGAAGTGGATTCCGTCSNNGCCGTCGGTGCTGATCAC (SEQ ID NO: 553) D192 nsa393R CTCGGTGAAGTGGATTCCSNNGACGCCGTCGGTGCTGAT (SEQ ID NO: 554) G193 nsa394R GGCCTCGGTGAAGTGGATSNNGTCGACGCCGTCGGTGCT (SEQ ID NO: 555) I194 nsa181R GTTGGCCTCGGTGAAGTGSNNTCCGTCGACGCCGTCGGT (SEQ ID NO: 556) H195 nsa396R ATTGTTGGCCTCGGTGAASNNGATTCCGTCGACGCCGTC (SEQ ID NO: 557) F196 nsa182R GCGATTGTTGGCCTCGGTSNNGTGGATTCCGTCGACGCC (SEQ ID NO: 558) T197 nsa398R ATCGCGATTGTTGGCCTCSNNGAAGTGGATTCCGTCGAC (SEQ ID NO: 559) E198 nsa399R GAGATCGCGATTGTTGGCSNNGGTGAAGTGGATTCCGTC (SEQ ID NO: 560) A199 nsa400R CCCGAGATCGCGATTGTTSNNCTCGGTGAAGTGGATTCC (SEQ ID NO: 561) N200 nsa401R CACCCCGAGATCGCGATTSNNGGCCTCGGTGAAGTGGAT (SEQ ID NO: 562) N201 nsa402R GGCCACCCCGAGATCGCGSNNGTTGGCCTCGGTGAAGTG (SEQ ID NO: 563) R202 nsa403R GAGGGCCACCCCGAGATCSNNATTGTTGGCCTCGGTGAA (SEQ ID NO: 564) D203 nsa404R CGCGAGGGCCACCCCGAGSNNGCGATTGTTGGCCTCGGT (SEQ ID NO: 565) L204 nsa405R TTCCGCGAGGGCCACCCCSNNATCGCGATTGTTGGCCTC (SEQ ID NO: 566) G205 nsa406R CTGTTCCGCGAGGGCCACSNNGAGATCGCGATTGTTGGC (SEQ ID NO: 567) V206 nsa407R CACCTGTTCCGCGAGGGCSNNCCCGAGATCGCGATTGTT (SEQ ID NO: 568) A207 nsa408R CTGCACCTGTTCCGCGAGSNNCACCCCGAGATCGCGATT (SEQ ID NO: 569) L208 nsa409R GCTCTGCACCTGTTCCGCSNNGGCCACCCCGAGATCGCG (SEQ ID NO: 570) A209 nsa410R CAGGCTCTGCACCTGTTCSNNGAGGGCCACCCCGAGATC (SEQ ID NO: 571) E210 nsa411R CAGCAGGCTCTGCACCTGSNNCGCGAGGGCCACCCCGAG (SEQ ID NO: 572) Q211 nsa412R TTACAGCAGGCTCTGCACSNNTTCCGCGAGGGCCACCCC (SEQ ID NO: 573) V212 nsa413R CTTTTACAGCAGGCTCTGSNNCTGTTCCGCGAGGGCCAC (SEQ ID NO: 574) Q213 nsa414R GCCCTTTTACAGCAGGCTSNNCACCTGTTCCGCGAGGGC (SEQ ID NO: 575) S214 nsa415R TTCGCCCTTTTACAGCAGSNNCTGCACCTGTTCCGCGAG (SEQ ID NO: 576) L215 nsa416R GAATTCGCCCTTTTACAGSNNGCTCTGCACCTGTTCCGC (SEQ ID NO: 577) L216 nsa417R GCAGAATTCGCCCTTTTASNNCAGGCTCTGCACCTGTTC (SEQ ID NO: 578)

QC Method to Create Site-Saturation Libraries

The QC reaction consisted of 40.25 μL of sterile distilled H₂O, 5 μL of PfuTurbo 10× buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL of forward primer (100 ngpL), 1.25 μL reverse primer (100 ngpL), 0.25 μL of pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 μL of PfuTurbo from the kit, for a total of 50 μL. The cycling conditions were 95° C. for 1 min, once, followed by 19-20 cycles of 95° C. for 30 to 45 sec, 55° C. for 1 min, and 68° C. for 5 to 8 min. To analyze the reaction, 5 μL of the reaction was run on a 0.8% E-gel (Invitrogen) upon completion. Next, Dpnl digestion was carried out twice sequentially, with 1 μL and 0.5 μL of enzyme at 37° C. for 2 to 8 hours. A negative control was carried out under similar conditions, but without any primers. Then, 1 μL of the DpnI-digested reaction product was transformed into 50 μL of one-shot TOP10 electrocompetent cells (Invitrogen) using a BioRad electroporator. Then, 300 μL of SOC provided with the TOP10 cells (Invitrogen) were added to the electroporated cells and incubated with shaking for 1 hour before plating on LA plates containing 10 ppm kanamycin. The plates were incubated at 37° C. overnight. After this incubation, 96 colonies from each of the libraries (i.e., each site) were inoculated in 200 μL of LB containing 10-50 ppm of kanamycin in 96-well microtiter plates. The plates were frozen at −80° C. after addition of glycerol to 20% final concentration, and they were used for high throughput sequencing at Genaissance with the M13F and M13R primers.

QCMS Method to Create Site-Saturation Libraries

The QCMS reaction consisted of 19.25 μL of sterile distilled H₂O, 2.5 μL of 10× buffer from the kit, 1 μL dNTPs from the kit, 1 μL of 5′ phosphorylated forward primer (100 ngpL), 0.25 μL of pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 μL of the enzyme blend from the kit for a total of 25 μL. The cycling conditions were 95° C. for 1 min once, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and 68° C. for 8 min. To analyze the reaction product, 5 μL of the reaction were run on a 0.8% E-gel (Invitrogen) upon completion. Next, Dpnl digestion was carried out twice sequentially, with 0.5 μL of enzyme at 37° C. for 2 to 8 hours. The controls, transformation, and sequencing was performed as for the QC method described above.

Details of Screening Plate Preparation

Using a sterilized stamping tool with 96 pins, the frozen clones from each sequenced library plate were stamped on to a large LA plate containing 10 ppm kanamycin. The plate was then incubated overnight at 37° C. Individual mutant clones each representing each one of the 19 substitutions (or as many that were obtained) were inoculated into a Costar 96-well plate containing 195 μL of LB made with 2 fold greater yeast extract and 10 ppm kanamycin. Each mutant clone for a given site was inoculated in quadruplicate. The plate was grown at 37° C. and 225 rpm shaking for 18 hrs in a humidified chamber. In a separate 96-well plate, 26 μL of BugBuster (Novagen) with DNase were added to each well. Next, 125 μL of the library clone cultures were added to the BugBuster-containing plate in corresponding wells and the plate was frozen at 80° C. The plate was thawed, frozen and thawed again before use of the lysates in the peracid formation and peracid hydrolysis assays described herein.

Combinatorial Libraries and Mutants

From the screening of the single site-saturation libraries, the important sites and substitutions were identified and combined in different combinatorial libraries. For example, libraries described in Table 8-3 were created using the following sites and substitutions:

L12C, Q, G T25S, G, P L53H, Q, G, S S54V, L, A, P, T, R A55G, T R67T, Q, N, G, E, L, F K97R V125S, G, R, A, P F154Y F196G

TABLE 8-3 Libraries Parent Library Description Template Method NSAA1 L12G S54(NNS) L12G QC NSAA2 S54V L12(NNS) S54V QC NSAA3 L12(NNS) S54(NNS) WT QCMS NSAB1 S54V T25(NNS) S54V QC NSAB2 S54V R67(NNS) S54V QC NSAB3 S54V V125(NNS) S54V QC NSAB4 L12I S54V T25(NNS) L12I S54V QC NSAB5 L12I S54V R67(NNS) L12I S54V QC NSAB6 L12I S54V V125(NNS) L12I S54V QC NSAC1 S54(NNS) R67(NNS) WT QCMS V125(NNS) NSAC2 43 primer library; 10 sites S54V QCMS (100 ng total primers) NSAC3 same as nsaC2 but 300 ng S54V QCMS total primers NSAC4 32 primer library, 8 sites S54V QCMS (100 ng total primers) NSAC5 same as nsaC4 but 300 ng S54V QCMS total primers NSAC6 8 primers, 7 substitutions, S54V QCMS 5 sites (100 ng total primers) NSAC7 same as nsaC6 but 300 ng S54V QCMS total primers *NNS indicates site-saturation library **All parent templates were derived from the pMSAT-NcoI plasmid and contained mutations at the indicated codons with in the M. smegmatis perhydrolase gene

-   -   *NNS indicates site-saturation library     -   **All parent templates were derived from the pMSAT-NcoI plasmid         and contained mutations at the indicated codons with in the M.         smegmatis perhydrolase gene

The QC or QCMS methods were used to create the combinations. The QC reaction was carried out as described above, with the exception being the template plasmid, which consisted of 0.25 μL of miniprep DNA of the L12G mutant, S54V mutant, or the L121S54V double mutant plasmid derived from pMSAT-NcoI. The QCMS reaction was also carried out as described above, with the exception of template and primers. In this case, 0.25 μL of the pMSAT-NcoI template were used for NSAC1 and NSAA3 or S54V template for NSAC2-C7 libraries. The NSAA3 and the NSAC1 libraries were made using 100 ng of each of the primers shown in the Table 8-4. The NSAC2, NSAC4, and NSAC6 libraries were made with a total of 100 ng of all primers (all primers being equimolar), and NSAC3, NSAC5, NSAC7 libraries were made with a total of 300 ng of all primers (all primers being approximately equimolar)

TABLE 8-4 Libraries Primer Libraries Name Primer Sequence NSAC1 S54NNS-FP gtgatcgaggagggactgnnsgcgcgcaccaccaacatc (SEQ ID NO: 579) NSAC1 R67NNS-FP acgaccccaccgatccgnnsctcaacggcgcgagctac (SEQ ID NO: 580) NSAC1 V125NNS- ctcaccagcgcgggcggcnnsggcaccacgtacccggca (SEQ ID NO: 581) FP NSAC2- L12C ctgtgtttcggtgattccTGCacctggggctgggtcccc (SEQ ID NO: 582) C5 NSAC2- L12Q ctgtgtttcggtgattccCAGacctggggctgggtcccc (SEQ ID NO: 583) C7 NSAC2- L12I ctgtgtttcggtgattccATCacctggggctgggtcccc (SEQ ID NO: 584) C5 NSAC2- L12M ctgtgtttcggtgattccATGacctggggctgggtcccc (SEQ ID NO: 585) C3 NSAC2- L12T ctgtgtttcggtgattccACGacctggggctgggtcccc (SEQ ID NO: 586) C3 NSA2- T25S gtcgaagacggggcacccAGCgagcggttcgcccccgac (SEQ ID NO: 587) C5 NSAC2- T25G gtcgaagacggggcacccGGCgagcggttcgcccccgac (SEQ ID NO: 588) C5 NSAC2- T25P gtcgaagacggggcacccCCGgagcggttcgcccccgac (SEQ ID NO: 589) C3 NSAC2- L53H gaggtgatcgaggagggaCACagcgcgcgcaccaccaac (SEQ ID NO: 590) C7 NSAC2- L53Q gaggtgatcgaggagggaCAGagcgcgcgcaccaccaac (SEQ ID NO: 591) C3 NSAC2- L53G gaggtgatcgaggagggaGGCagcgcgcgcaccaccaac (SEQ ID NO: 592) C3 NSAC2- L53S gaggtgatcgaggagggaAGCagcgcgcgcaccaccaac (SEQ ID NO: 593) C3 NSAC2- L53HS54V gaggtgatcgaggagggaCACGTGgcgcgcaccaccaac (SEQ ID C7 NO: 594) NSAC2- L53QS54V gaggtgatcgaggagggaCAGGTGgcgcgcaccaccaac (SEQ ID C3 NO: 595) NSAC2- L53GS54V gaggtgatcgaggagggaGGCGTGgcgcgcaccaccaac (SEQ ID C3 NO: 596) NSAC2- L53SS54V gaggtgatcgaggagggaAGCGTGgcgcgcaccaccaac (SEQ ID C3 NO: 597) NSAC2- S54V gtgatcgaggagggactgGTGgcgcgcaccaccaacatc (SEQ ID NO: 598) C7 NSAC2- S54L gtgatcgaggagggactgCTGgcgcgcaccaccaacatc (SEQ ID NO: 599) C5 NSAC2- A55G atcgaggagggactgagcGGCcgcaccaccaacatcgac (SEQ ID NO: 600) C5 NSAC2- A55T atcgaggagggactgagcACGcgcaccaccaacatcgac (SEQ ID NO: 601) C5 NSAC2- A55GS54V atcgaggagggactgGTGGGCcgcaccaccaacatcgac (SEQ ID NO: 602) C5 NSAC2- A55TS54V atcgaggagggactgGTGACGcgcaccaccaacatcgac (SEQ ID NO: 603) C5 NSAC2- R67T gacgaccccaccgatccgACGctcaacggcgcgagctac (SEQ ID NO: 604) C5 NSAC2- R67Q gacgaccccaccgatccgCAGctcaacggcgcgagctac (SEQ ID NO: 605) C5 NSAC2- R67N gacgaccccaccgatccgAACctcaacggcgcgagctac (SEQ ID NO: 606) C7 NSAC2- K97R ctgggcaccaacgacaccCGCgcctacttccggcgcacc (SEQ ID NO: 607) C5 NSAC2- V125S ctcaccagcgcgggcggcAGCggcaccacgtacccggca (SEQ ID NO: 608) C5 NSAC2- V125G ctcaccagcgcgggcggcGGCggcaccacgtacccggca (SEQ ID NO: 609) C7 NSAC2- V125R ctcaccagcgcgggcggcCGCggcaccacgtacccggca (SEQ ID NO: 610) C5 NSAC2- V125A ctcaccagcgcgggcggcGCGggcaccacgtacccggca (SEQ ID NO: 611) C5 NSAC2- V125P ctcaccagcgcgggcggcCCGggcaccacgtacccggca (SEQ ID NO: 612) C5 NSAC2- F154Y ccctggttccagttgatcTACgagggcggcgagcagaag (SEQ ID NO: 613) C3 NSAC2- F196G ggcgtcgacggaatccacGGCaccgaggccaacaatcgc (SEQ ID NO: 614) C3 NSAC2- R67G-re gacgaccccaccgatccgGGCctcaacggcgcgagctac (SEQ ID NO: 615) C7 NSAC2- R67E-re gacgaccccaccgatccgGAGctcaacggcgcgagctac (SEQ ID NO: 616) C5 NSAC2- R67F-re gacgaccccaccgatccgTTCctcaacggcgcgagctac (SEQ ID NO: 617) C5 NSAC2- R67L-re gacgaccccaccgatccgCTGctcaacggcgcgagctac (SEQ ID NO: 618) C5 NSAC2- S54P gtgatcgaggagggactgCCGgcgcgcaccaccaacatc (SEQ ID NO: 619) C5 NSAC2- S54R gtgatcgaggagggactgCGCgcgcgcaccaccaacatc (SEQ ID NO: 620) C5 NSAC2- S54G gtgatcgaggagggactgGGCgcgcgcaccaccaacatc (SEQ ID NO: 621) C5 NSAC2- S54T gtgatcgaggagggactgACGgcgcgcaccaccaacatc (SEQ ID NO: 622) C5 NSAC2- S54I gtgatcgaggagggactgATCgcgcgcaccaccaacatc (SEQ ID NO: 623) C7 NSAC2- S54K gtgatcgaggagggactgAAGgcgcgcaccaccaacatc (SEQ ID NO: 624) C5

Screening of Combinatorial Libraries and Mutants

For each of the NSAB1-B6 libraries, a 96-well plate full of clones was first sequenced. Once the sequencing results were analyzed, the mutants obtained for each library were inoculated in quadruplicate, similar to the site-saturation libraries described above. For the NSAC1-C7 libraries, 96 colonies perplatelibrary were initially inoculated, and each plate was screened without sequencing. Upon screening, some libraries looked better than others. Several plates for each of the NSAC1, C2, C4, C6 libraries were screened. The “winners” from these single isolate screening plates were then streaked out for singles or directly screened in quadruplicate just like the site-saturation libraries (i.e., as described above). Only the “winners” identified were sequenced.

Example 9 Improved Properties of Multiply Mutated Perhydrolase Variants

In this Example, experiments conducted to assess the properties of multiply-mutated perhydrolase variants are described. In these experiments, combinatorial mutants obtained from combinatorial libraries were tested in their performance in perhydrolysis, peracid hydrolysis and perhydrolysis to hydrolysis ratio. These parameters were measured in the HPLC or ABTS assays described in Example 2, above. Combinatorial variants tested were:

L12I S54V, L12M S54T, L12T S54V, L12Q T25S S54V, L53H S54V, S54P V125R, S54V V125G, S54V F196G, S54V K97R V125G, and A55G R67T K97R V125G,

As is indicated in Table 9-1 below, all of these variants were better than wild type enzyme in at least one of the properties of interest.

TABLE 9-1 Results for Multiple Variants Fold-Improvement in Property Multiple Variant Perhydrolysis Peracid Hydrolysis Ratio L12I S54V 2 2.5 L12M S54T 1.6 3 L12T S54V 1.5 2.5 L12Q T25S S54V 4 to 5 L53H S54V 2 4 to 5 S54P V125R 4 S54V V125G 2 4 S54V F196G 2 S54V K97R V125G 2 A55G R67T K97R 1.6 4 to 5 V125G

Example 10 PAF and PAD Assays of Perhydrolase Variants

In this Example, assay results for PAF and PAD testing of perhydrolase variants are provided. The tests were conducted as described in Example 1, above. In addition, Tables are provided in which the protein expression of the variant was greater than wild-type under the same culture conditions (described herein). These results are indicated as the “protein performance index.” Thus, a number greater than “1” in the protein performance index indicates that more protein was made for the particular variant than the wild-type. In the following Tables, “WT” indicates the wild-type amino acid residue; “Pos” indicates the position in the amino acid sequence; “Mut.” and “Var” indicate the amino acid residue substituted at that particular position; “prot.” indicates “protein; and “Perf. Ind” indicates the performance index.

TABLE 10-1 PAF Assay Results PAF WT/Pos/ Perf. Position Mutation Variant Ind. 3 K003Y Y 1.058244 3 K003I I 1.053242 3 K003L L 1.038686 3 K003T T 1.009071 3 K003H H 1.00528 4 R004Q Q 1.025332 5 I005T T 1.12089 5 I005S S 1.023576 6 L006V V 1.072388 6 L006I I 1.066182 6 L006T T 1.062078 7 C007K K 2.687956 7 C007Y Y 2.08507 7 C007I I 1.758096 7 C007H H 1.731475 7 C007A A 1.423943 7 C007G G 1.393781 7 C007M M 1.126028 10 D010L L 3.97014 10 D010W W 3.179778 10 D010K K 2.133852 10 D010Y Y 1.508981 10 D010T T 1.473387 10 D010I I 1.281927 12 L012Q Q 2.651732 12 L012C C 2.289224 12 L012A A 1.100171 15 G015A A 1.543799 15 G015S S 1.05273 17 V017G G 1.173641 17 V017R R 1.09735 17 V017A A 1.012116 18 P018Y Y 1.332844 18 P018N N 1.331062 18 P018C C 1.261104 18 P018E E 1.217708 18 P018V V 1.185736 18 P018R R 1.16328 18 P018Q Q 1.124133 18 P018H H 1.120443 18 P018G G 1.068272 19 V019G G 1.317001 19 V019S S 1.235759 19 V019R R 1.025471 19 V019L L 1.002833 21 D021K K 1.062138 21 D021W W 1.040173 22 G022A A 1.554264 22 G022T T 1.032118 22 G022S S 1.022133 25 T025G G 1.857878 25 T025S S 1.59954 25 T025A A 1.327579 25 T025I I 1.019417 26 E026M M 2.002044 26 E026A A 1.927099 26 E026R R 1.484814 26 E026K K 1.464368 26 E026T T 1.441939 26 E026C C 1.403045 26 E026V V 1.392881 26 E026N N 1.366419 26 E026H H 1.329562 26 E026L L 1.295378 26 E026G G 1.283477 26 E026S S 1.271403 26 E026W W 1.251752 27 R027K K 1.215697 28 F028M M 1.331874 28 F028A A 1.269493 28 F028W W 1.156698 28 F028L L 1.08849 28 F028S S 1.046063 29 A029W W 1.912244 29 A029V V 1.799733 29 A029R R 1.757225 29 A029Y Y 1.697554 29 A029G G 1.595061 29 A029S S 1.486877 29 A029T T 1.424584 29 A029E E 1.115768 29 A029C C 1.07522 30 P030K K 1.207673 30 P030R R 1.164892 30 P030V V 1.063047 30 P030T T 1.05383 30 P030A A 1.045476 30 P030S S 1.031747 30 P030Q Q 1.013468 30 P030H H 1.012332 30 P030E E 1.006761 31 D031W W 1.834044 31 D031L L 1.810564 31 D031T T 1.450556 31 D031G G 1.441703 31 D031F F 1.438268 31 D031N N 1.339422 31 D031V V 1.280091 31 D031A A 1.240923 31 D031R R 1.222181 31 D031S S 1.152736 31 D031E E 1.132795 31 D031Q Q 1.069797 32 V032K K 1.08606 32 V032R R 1.045435 33 R033S S 1.000491 36 G036I I 1.320156 36 G036K K 1.265563 36 G036L L 1.237473 38 L038L L 6.528092 38 L038V V 5.735873 38 L038C C 4.182031 38 L038K K 4.135067 38 L038A A 3.844719 38 L038S S 2.467764 40 Q040K K 2.613726 40 Q040I I 2.576806 40 Q040W W 2.394926 40 Q040L L 2.144687 40 Q040T T 2.006487 40 Q040R R 1.885154 40 Q040Y Y 1.825366 40 Q040G G 1.785768 40 Q040S S 1.565973 40 Q040N N 1.528677 40 Q040D D 1.16151 40 Q040E E 1.075259 41 Q041K K 1.381385 41 Q041R R 1.190317 41 Q041W W 1.141041 41 Q041H H 1.123719 41 Q041S S 1.107641 41 Q041Y Y 1.091652 41 Q041V V 1.070265 41 Q041A A 1.032945 41 Q041L L 1.000416 42 L042K K 2.463086 42 L042W W 2.056507 42 L042H H 1.917245 42 L042R R 1.378137 42 L042G G 1.172748 42 L042T T 1.079826 42 L042F F 1.072948 43 G043A A 1.49082 43 G043C C 1.47701 43 G043K K 1.424919 43 G043M M 1.371202 43 G043Y Y 1.262703 43 G043E E 1.250311 43 G043L L 1.216516 43 G043R R 1.215829 43 G043S S 1.178103 43 G043H H 1.169457 43 G043P P 1.080176 44 A044F F 2.84399 44 A044V V 2.133682 44 A044C C 1.796096 44 A044L L 1.607918 44 A044W W 1.395243 44 A044M M 1.199028 45 D045K K 1.342858 45 D045T T 1.268367 45 D045R R 1.158768 45 D045W W 1.145157 45 D045S S 1.133098 45 D045G G 1.12761 45 D045H H 1.127539 45 D045F F 1.11152 45 D045L L 1.054441 45 D045V V 1.050576 45 D045Q Q 1.04498 45 D045A A 1.037993 46 F046E E 1.247552 46 F046D D 1.174794 46 F046G G 1.016913 46 F046K K 1.003326 47 E047R R 2.448525 47 E047T T 1.960505 47 E047P P 1.361173 47 E047S S 1.278809 47 E047H H 1.266229 47 E047G G 1.197541 47 E047K K 1.19183 47 E047F F 1.092281 47 E047I I 1.030029 49 I049G G 1.342918 49 I049H H 1.265204 49 I049S S 1.238211 49 I049K K 1.230871 49 I049V V 1.203314 49 I049L L 1.136805 49 I049Y Y 1.068104 49 I049R R 1.052285 49 I049E E 1.015762 49 I049M M 1.00526 50 E050L L 1.191901 50 E050M M 1.178039 50 E050A A 1.124087 51 E051V V 1.471315 51 E051A A 1.279983 51 E051G G 1.217963 51 E051T T 1.182792 51 E051L L 1.112889 51 E051I I 1.072835 53 L053H H 5.05321 53 L053Q Q 1.480206 53 L053G G 1.317357 53 L053S S 1.161011 53 L053T T 1.019146 54 S054P P 5.198689 54 S054I I 4.775938 54 S054V V 4.722033 54 S054A A 3.455902 54 S054R R 3.375793 54 S054L L 2.015828 54 S054T T 1.459971 54 S054K K 1.438715 54 S054G G 1.429605 54 S054C C 1.259773 54 S054Q Q 1.03365 55 A055G G 1.694814 55 A055T T 1.692885 57 T057S S 1.633613 57 T057R R 1.605072 57 T057V V 1.281788 57 T057I I 1.189062 59 N059W W 1.035044 59 N059R R 1.002315 60 I060H H 1.02415 60 I060R R 1.003947 61 D061H H 1.439407 61 D061S S 1.259714 61 D061R R 1.105425 61 D061I I 1.076937 61 D061F F 1.00566 62 D062E E 1.019293 63 P063G G 1.709657 63 P063T T 1.499483 63 P063M M 1.460336 63 P063S S 1.416192 63 P063K K 1.404615 63 P063A A 1.347541 63 P063Y Y 1.346046 63 P063W W 1.34587 63 P063V V 1.313631 63 P063R R 1.310696 63 P063F F 1.246299 63 P063L L 1.146416 63 P063Q Q 1.093179 64 T064G G 1.234467 64 T064S S 1.114348 65 D065A A 1.312312 65 D065S S 1.166849 65 D065H H 1.096335 66 P066R R 1.846257 66 P066V V 1.828926 66 P066H H 1.589631 66 P066I I 1.588219 66 P066G G 1.499901 66 P066Q Q 1.463705 66 P066T T 1.410091 66 P066S S 1.390845 66 P066Y Y 1.330685 66 P066L L 1.137635 66 P066N N 1.122261 67 R067N N 1.580401 67 R067G G 1.390129 67 R067T T 1.284643 67 R067F F 1.25763 67 R067L L 1.203316 67 R067Q Q 1.164899 67 R067W W 1.066028 67 R067E E 1.044676 67 R067P P 1.012761 68 L068E E 1.435218 68 L068W W 1.209193 68 L068I I 1.125898 68 L068G G 1.092454 68 L068V V 1.088042 68 L068H H 1.051612 68 L068T T 1.032331 69 N069V V 1.989028 69 N069K K 1.71908 69 N069R R 1.493163 69 N069I I 1.469946 69 N069H H 1.357968 69 N069T T 1.351305 69 N069L L 1.299547 69 N069S S 1.205171 69 N069G G 1.19653 69 N069Q Q 1.074622 69 N069W W 1.049602 69 N069C C 1.048373 71 A071S S 1.751794 71 A071T T 1.700442 71 A071H H 1.697558 71 A071G G 1.58881 71 A071I I 1.507841 71 A071E E 1.445699 71 A071K K 1.441146 71 A071R R 1.401499 71 A071N N 1.232241 71 A071L L 1.231991 71 A071F F 1.127538 71 A071C C 1.00977 72 S072L L 1.257945 72 S072H H 1.208899 72 S072G G 1.198197 72 S072T T 1.10065 72 S072V V 1.080089 72 S072Y Y 1.066178 73 Y073R R 1.2555 73 Y073Q Q 1.23429 73 Y073S S 1.165683 73 Y073K K 1.070678 76 S076P P 1.229172 77 C077T T 1.120603 77 C077V V 1.052586 77 C077G G 1.013806 78 L078G G 4.975852 78 L078H H 4.824004 78 L078E E 3.007159 78 L078N N 2.683604 78 L078T T 1.867711 78 L078Q Q 1.726942 78 L078V V 1.534239 78 L078I I 1.434206 78 L078Y Y 1.387889 79 A079H H 1.927914 79 A079L L 1.796126 79 A0791 I 1.592463 79 A079M M 1.499635 79 A079N N 1.475806 79 A079Q Q 1.472484 79 A079R R 1.465943 79 A079W W 1.270538 79 A079T T 1.169146 79 A079E E 1.123457 80 T080C C 1.310752 80 T080V V 1.230659 80 T080G G 1.160318 80 T080A A 1.000722 82 L082P P 1.456374 82 L082G G 1.379439 82 L082R R 1.339485 82 L082H H 1.332844 82 L082K K 1.1909 82 L082T T 1.17992 82 L082I I 1.171013 82 L082S S 1.153417 82 L082V V 1.019854 83 P083K K 1.369406 83 P083G G 1.313431 83 P083H H 1.265876 83 P083R R 1.194464 83 P083S S 1.171208 84 L084K K 1.099089 84 L084H H 1.008187 85 D085Q Q 3.093245 85 D085R R 2.379647 85 D085S S 2.284009 85 D085H H 1.548556 85 D085N N 1.539497 85 D085G G 1.413812 85 D085T T 1.329395 85 D085E E 1.117228 85 D085F F 1.008028 86 L086A A 1.376284 86 L086C C 1.156625 86 L086G G 1.145834 95 D095E E 2.044825 96 T096S S 1.044425 97 K097R R 2.798748 97 K097Q Q 1.136975 100 F100W W 1.082799 100 F100E E 1.0116 101 R101K K 1.244945 103 T103W W 1.261503 103 T103Y Y 1.193299 103 T103G G 1.113343 103 T103K K 1.093573 103 T103I I 1.076338 103 T103L L 1.050734 104 P104H H 2.837034 104 P104T T 2.696977 104 P104G G 2.672719 104 P104V V 2.585315 104 P104S S 2.481687 104 P104I I 2.431309 104 P104W W 2.051785 104 P104C C 1.951282 104 P104E E 1.837373 104 P104F F 1.785718 104 P104N N 1.624722 104 P104R R 1.618032 104 P104Q Q 1.343174 104 P104M M 1.093185 105 L105P P 1.713219 105 L105C C 1.557999 105 L105F F 1.295759 105 L105W W 1.283998 105 L105G G 1.078743 106 D106K K 1.278457 106 D106L L 1.198148 106 D106G G 1.178297 106 D106H H 1.090134 106 D106E E 1.084931 106 D106T T 1.061622 106 D106I I 1.036191 106 D106F F 1.021513 106 D106C C 1.005553 107 I107E E 2.551108 107 I107S S 2.044692 107 I107N N 1.810584 107 I107G G 1.764761 107 I107V V 1.001703 108 A108L L 1.407382 108 A108T T 1.050964 109 L109N N 1.523277 109 L109W W 1.296964 109 L109Q Q 1.182653 109 L109Y Y 1.155328 109 L109I I 1.053129 109 L109D D 1.003394 111 M111K K 1.977248 111 M111I I 1.949343 111 M111L L 1.546317 111 M111T T 1.489808 111 M111F F 1.467344 111 M111V V 1.466478 111 M111Y Y 1.42589 111 M111S S 1.031939 112 S112L L 1.027928 112 S112H H 1.001485 113 V113L L 1.503622 113 V113H H 1.339003 113 V113K K 1.192607 113 V113R R 1.133751 113 V113Y Y 1.113256 113 V113F F 1.045057 113 V113Q Q 1.032496 115 V115W W 1.234 115 V115T T 1.145757 115 V115L L 1.117398 115 V115G G 1.089596 115 V115I I 1.050387 115 V115Y Y 1.032052 116 T116G G 1.095496 116 T116A A 1.006702 117 Q117H H 2.327857 117 Q117T T 2.233854 117 Q117Y Y 2.227983 117 Q117W W 2.155359 117 Q117V V 2.154646 117 Q117G G 2.080223 117 Q117A A 2.048752 117 Q117S S 1.949232 117 Q117F F 1.573776 117 Q117R R 1.564466 117 Q117M M 1.541944 117 Q117E E 1.145341 118 V118Y Y 1.25067 118 V118K K 1.125917 118 V118G G 1.083422 120 T120S S 1.089798 121 S121L L 1.348931 121 S121W W 1.333741 121 S121R R 1.25879 121 S121K K 1.241105 121 S121G G 1.204547 121 S121C C 1.177769 121 S121N N 1.143954 121 S121T T 1.132507 121 S121A A 1.120633 121 S121V V 1.120454 122 A122H H 1.137861 122 A122I I 1.133601 122 A122T T 1.083131 122 A122K K 1.082552 122 A122V V 1.041449 122 A122S S 1.031411 124 G124L L 1.91642 124 G124I I 1.853337 124 G124T T 1.63716 124 G124H H 1.588068 124 G124V V 1.441979 124 G124F F 1.320782 124 G124S S 1.269245 124 G124Y Y 1.234423 124 G124R R 1.144212 124 G124Q Q 1.123498 125 V125G G 2.948291 125 V125S S 1.942881 125 V125A A 1.689696 125 V125P P 1.50166 125 V125R R 1.301534 125 V125D D 1.238852 125 V125Y Y 1.080394 125 V125I I 1.010779 126 G126T T 1.577938 126 G126P P 1.171092 126 G126L L 1.169527 127 T127H H 1.57251 127 T127V V 1.073821 127 T127I I 1.063668 127 T127S S 1.046984 128 T128L L 1.064623 128 T128K K 1.062947 148 P148V V 2.426937 148 P148K K 1.786508 148 P148L L 1.638438 148 P148A A 1.637334 148 P148R R 1.509086 148 P148T T 1.501359 148 P148Y Y 1.459512 148 P148S S 1.45564 148 P148E E 1.417449 148 P148F F 1.367568 148 P148Q Q 1.334517 148 P148D D 1.030185 150 F150L L 1.290835 150 F150E E 1.228159 153 I153K K 1.618543 153 I153H H 1.464262 153 I153T T 1.271928 153 I153L L 1.270149 153 I153F F 1.227821 153 I153A A 1.194659 154 F154Y Y 1.323693 196 F196H H 1.774774 196 F196L L 1.768072 196 F196C C 1.738263 196 F196M M 1.647608 196 F196G G 1.590716 196 F196S S 1.577837 196 F196Y Y 1.414589 196 F196V V 1.395387 196 F196I I 1.320955 196 F196W W 1.014435

The following Table provides variants with PAF results that were better than those observed for wild-type M. smegmatis perhydrolase. In this Table, the middle column indicates the amino acid residue in the wild-type perhydrolase (WT), followed by the position number and the variant amino acid in that position (Var).

TABLE 10-2 Variants with PAF Values Better Than Wild-Type WT/Pos./ Peracid formation Pos Var relative to WT 2 A002W 1.75 2 A002D 1.30 2 A002F 1.24 2 A002I 1.18 2 A002G 1.15 2 A002S 1.01 3 K003Y 1.06 3 K003I 1.05 3 K003L 1.04 3 K003T 1.01 3 K003H 1.01 4 R004Q 1.03 5 I005T 1.12 5 I005S 1.02 6 L006V 1.07 6 L006I 1.07 6 L006T 1.06 7 C007K 2.69 7 C007Y 2.09 7 C007I 1.76 7 C007H 1.73 7 C007A 1.42 7 C007G 1.39 7 C007M 1.13 8 F008R 1.43 8 F008V 1.18 8 F008G 1.09 8 F008H 1.02 10 D010L 3.97 10 D010W 3.18 10 D010K 2.13 10 D010Y 1.51 10 D010T 1.47 10 D010I 1.28 12 L012Q 2.65 12 L012C 2.29 12 L012A 1.10 15 G015A 1.54 15 G015S 1.05 17 V017G 1.17 17 V017R 1.10 17 V017A 1.01 18 P018Y 1.33 18 P018N 1.33 18 P018C 1.26 18 P018E 1.22 18 P018V 1.19 18 P018R 1.16 18 P018Q 1.12 18 P018H 1.12 18 P018G 1.07 19 V019G 1.32 19 V019S 1.24 19 V019R 1.03 19 V019L 1.00 20 E020W 2.94 20 E020G 2.36 20 E020T 2.22 20 E020L 2.20 20 E020H 2.17 20 E020V 2.11 20 E020S 2.01 20 E020C 1.57 20 E020N 1.40 20 E020A 1.29 20 E020Q 1.27 21 D021K 1.58 21 D021W 1.55 21 D021L 1.46 21 D021A 1.46 21 D021G 1.37 21 D021Y 1.30 21 D021F 1.30 21 D021S 1.24 22 G022A 1.55 22 G022T 1.03 22 G022S 1.02 25 T025G 1.86 25 T025S 1.60 25 T025A 1.33 25 T025I 1.02 26 E026M 2.00 26 E026A 1.93 26 E026R 1.48 26 E026K 1.46 26 E026T 1.44 26 E026C 1.40 26 E026V 1.39 26 E026N 1.37 26 E026H 1.33 26 E026L 1.30 26 E026G 1.28 26 E026S 1.27 26 E026W 1.25 27 R027K 1.22 28 F028M 1.33 28 F028A 1.27 28 F028W 1.16 28 F028L 1.09 28 F028S 1.05 29 A029W 1.91 29 A029V 1.80 29 A029R 1.76 29 A029Y 1.70 29 A029G 1.60 29 A029S 1.49 29 A029T 1.42 29 A029E 1.12 29 A029C 1.08 30 P030K 1.21 30 P030R 1.16 30 P030V 1.06 30 P030T 1.05 30 P030A 1.05 30 P030S 1.03 30 P030Q 1.01 30 P030H 1.01 30 P030E 1.01 31 D031W 1.83 31 D031L 1.81 31 D031T 1.45 31 D031G 1.44 31 D031F 1.44 31 D031N 1.34 31 D031V 1.28 31 D031A 1.24 31 D031R 1.22 31 D031S 1.15 31 D031E 1.13 31 D031Q 1.07 32 V032K 1.09 32 V032R 1.05 33 R033S 1.00 36 G036I 1.32 36 G036K 1.27 36 G036L 1.24 37 V037S 1.40 37 V037I 1.26 37 V037A 1.25 37 V037H 1.21 37 V037L 1.16 37 V037C 1.09 37 V037T 1.05 39 A039L 1.43 39 A039K 1.36 39 A039Y 1.36 39 A039I 1.26 39 A039T 1.26 39 A039W 1.23 39 A039V 1.21 39 A039G 1.17 39 A039R 1.17 39 A039E 1.09 40 Q040K 2.61 40 Q040I 2.58 40 Q040W 2.39 40 Q040L 2.14 40 Q040T 2.01 40 Q040R 1.89 40 Q040Y 1.83 40 Q040G 1.79 40 Q040S 1.57 40 Q040N 1.53 40 Q040D 1.16 40 Q040E 1.08 41 Q041K 1.38 41 Q041R 1.19 41 Q041W 1.14 41 Q041H 1.12 41 Q041S 1.11 41 Q041Y 1.09 41 Q041V 1.07 41 Q041A 1.03 41 Q041L 1.00 42 L042K 2.46 42 L042W 2.06 42 L042H 1.92 42 L042R 1.38 42 L042G 1.17 42 L042T 1.08 42 L042F 1.07 43 G043A 1.49 43 G043C 1.48 43 G043K 1.42 43 G043M 1.37 43 G043Y 1.26 43 G043E 1.25 43 G043L 1.22 43 G043R 1.22 43 G043S 1.18 43 G043H 1.17 43 G043P 1.08 44 A044F 2.84 44 A044V 2.13 44 A044C 1.80 44 A044L 1.61 44 A044W 1.40 44 A044M 1.20 45 D045K 1.34 45 D045T 1.27 45 D045R 1.16 45 D045W 1.15 45 D045S 1.13 45 D045G 1.13 45 D045H 1.13 45 D045F 1.11 45 D045L 1.05 45 D045V 1.05 45 D045Q 1.04 45 D045A 1.04 46 F046E 1.25 46 F046D 1.17 46 F046G 1.02 46 F046K 1.00 47 E047R 2.45 47 E047T 1.96 47 E047P 1.36 47 E047S 1.28 47 E047H 1.27 47 E047G 1.20 47 E047K 1.19 47 E047F 1.09 47 E047I 1.03 49 I049G 1.34 49 I049H 1.27 49 I049S 1.24 49 I049K 1.23 49 I049V 1.20 49 I049L 1.14 49 I049Y 1.07 49 I049R 1.05 49 I049E 1.02 49 I049M 1.01 50 E050L 1.19 50 E050M 1.18 50 E050A 1.12 51 E051V 1.47 51 E051A 1.28 51 E051G 1.22 51 E051T 1.18 51 E051L 1.11 51 E051I 1.07 53 L053H 5.05 53 L053Q 1.48 53 L053G 1.32 53 L053S 1.16 53 L053T 1.02 54 S054P 5.20 54 S054I 4.78 54 S054V 4.72 54 S054A 3.46 54 S054R 3.38 54 S054L 2.02 54 S054T 1.46 54 S054K 1.44 54 S054G 1.43 54 S054C 1.26 54 S054Q 1.03 55 A055G 1.69 55 A055T 1.69 57 T057S 1.63 57 T057R 1.61 57 T057V 1.28 57 T057I 1.19 59 N059W 1.13 59 N059R 1.09 59 N059T 1.07 59 N059S 1.06 59 N059Q 1.02 60 I060H 1.02 60 I060R 1.00 61 D061H 1.44 61 D061S 1.26 61 D061R 1.11 61 D061I 1.08 61 D061F 1.01 62 D062E 1.02 63 P063G 1.71 63 P063T 1.50 63 P063M 1.46 63 P063S 1.42 63 P063K 1.40 63 P063A 1.35 63 P063Y 1.35 63 P063W 1.35 63 P063V 1.31 63 P063R 1.31 63 P063F 1.25 63 P063L 1.15 63 P063Q 1.09 64 T064G 1.23 64 T064S 1.11 65 D065A 1.31 65 D065S 1.17 65 D065H 1.10 66 P066R 1.85 66 P066V 1.83 66 P066H 1.59 66 P066I 1.59 66 P066G 1.50 66 P066Q 1.46 66 P066T 1.41 66 P066S 1.39 66 P066Y 1.33 66 P066L 1.14 66 P066N 1.12 67 R067N 1.58 67 R067G 1.39 67 R067T 1.28 67 R067F 1.26 67 R067L 1.20 67 R067Q 1.16 67 R067W 1.07 67 R067E 1.04 67 R067P 1.01 68 L068E 1.44 68 L068W 1.21 68 L068I 1.13 68 L068G 1.09 68 L068V 1.09 68 L068H 1.05 68 L068T 1.03 69 N069V 1.99 69 N069K 1.72 69 N069R 1.49 69 N069I 1.47 69 N069H 1.36 69 N069T 1.35 69 N069L 1.30 69 N069S 1.21 69 N069G 1.20 69 N069Q 1.07 69 N069W 1.05 69 N069C 1.05 71 A071S 1.75 71 A071T 1.70 71 A071H 1.70 71 A071G 1.59 71 A071I 1.51 71 A071E 1.45 71 A071K 1.44 71 A071R 1.40 71 A071N 1.23 71 A071L 1.23 71 A071F 1.13 71 A071C 1.01 72 S072L 1.26 72 S072H 1.21 72 S072G 1.20 72 S072T 1.10 72 S072V 1.08 72 S072Y 1.07 73 Y073R 1.26 73 Y073Q 1.23 73 Y073S 1.17 73 Y073K 1.07 74 L074S 2.72 74 L074G 1.95 74 L074W 1.38 75 P075R 1.60 75 P075S 1.39 75 P075T 1.28 75 P075Q 1.21 75 P075G 1.16 75 P075H 1.05 75 P075W 1.04 76 S076P 1.23 77 C077T 1.12 77 C077V 1.05 77 C077G 1.01 78 L078G 4.98 78 L078H 4.82 78 L078E 3.01 78 L078N 2.68 78 L078T 1.87 78 L078Q 1.73 78 L078V 1.53 78 L078I 1.43 78 L078Y 1.39 79 A079H 1.93 79 A079L 1.80 79 A079I 1.59 79 A079M 1.50 79 A079N 1.48 79 A079Q 1.47 79 A079R 1.47 79 A079W 1.27 79 A079T 1.17 79 A079E 1.12 80 T080C 1.31 80 T080V 1.23 80 T080G 1.16 80 T080A 1.00 81 H081K 1.52 81 H081L 1.23 81 H081N 1.17 81 H081G 1.17 81 H081A 1.15 81 H081C 1.13 81 H081W 1.13 81 H081V 1.10 81 H081F 1.10 81 H081S 1.04 82 L082P 1.46 82 L082G 1.38 82 L082R 1.34 82 L082H 1.33 82 L082K 1.19 82 L082T 1.18 82 L082I 1.17 82 L082S 1.15 82 L082V 1.02 83 P083K 1.37 83 P083G 1.31 83 P083H 1.27 83 P083R 1.19 83 P083S 1.17 84 L084K 1.10 84 L084H 1.01 85 D085Q 3.09 85 D085R 2.38 85 D085S 2.28 85 D085H 1.55 85 D085N 1.54 85 D085G 1.41 85 D085T 1.33 85 D085E 1.12 85 D085F 1.01 86 L086A 1.38 86 L086C 1.16 86 L086G 1.15 88 I088H 1.20 88 I088T 1.03 88 I088G 1.01 90 M090T 1.27 90 M090I 1.13 90 M090V 1.08 90 M090S 1.06 90 M090L 1.02 91 L091G 1.21 91 L091T 1.06 92 G092V 1.49 92 G092S 1.26 93 T093Y 5.26 93 T093F 3.52 93 T093A 1.38 93 T093C 1.08 95 D095E 2.04 96 T096S 1.04 97 K097R 2.80 97 K097Q 1.14 98 A098L 2.22 98 A098H 2.09 98 A098I 2.05 98 A098Y 2.02 98 A098S 1.73 98 A098T 1.72 98 A098G 1.57 98 A098C 1.30 98 A098N 1.24 98 A098D 1.11 98 A098P 1.10 100 F100W 1.08 100 F100E 1.01 101 R101K 1.24 103 T103W 1.26 103 T103Y 1.19 103 T103G 1.11 103 T103K 1.09 103 T103I 1.08 103 T103L 1.05 104 P104H 2.84 104 P104T 2.70 104 P104G 2.67 104 P104V 2.59 104 P104S 2.48 104 P104I 2.43 104 P104W 2.05 104 P104C 1.95 104 P104E 1.84 104 P104F 1.79 104 P104N 1.62 104 P104R 1.62 104 P104Q 1.34 104 P104M 1.09 105 L105P 1.71 105 L105C 1.56 105 L105F 1.30 105 L105W 1.28 105 L105G 1.08 106 D106K 1.28 106 D106L 1.20 106 D106G 1.18 106 D106H 1.09 106 D106E 1.08 106 D106T 1.06 106 D106I 1.04 106 D106F 1.02 106 D106C 1.01 107 I107E 2.55 107 I107S 2.04 107 I107N 1.81 107 I107G 1.76 107 I107V 1.00 108 A108L 1.41 108 A108T 1.05 109 L109N 1.52 109 L109W 1.30 109 L109Q 1.18 109 L109Y 1.16 109 L109I 1.05 109 L109D 1.00 111 M111K 1.98 111 M111I 1.95 111 M111L 1.55 111 M111T 1.49 111 M111F 1.47 111 M111V 1.47 111 M111Y 1.43 111 M111S 1.03 112 S112L 1.03 112 S112H 1.00 113 V113L 1.50 113 V113H 1.34 113 V113K 1.19 113 V113R 1.13 113 V113Y 1.11 113 V113F 1.05 113 V113Q 1.03 115 V115W 1.23 115 V115T 1.15 115 V115L 1.12 115 V115G 1.09 115 V115I 1.05 115 V115Y 1.03 116 T116G 1.10 116 T116A 1.01 117 Q117H 2.33 117 Q117T 2.23 117 Q117Y 2.23 117 Q117W 2.16 117 Q117V 2.15 117 Q117G 2.08 117 Q117A 2.05 117 Q117S 1.95 117 Q117F 1.57 117 Q117R 1.56 117 Q117M 1.54 117 Q117E 1.15 118 V118Y 1.25 118 V118K 1.13 118 V118G 1.08 120 T120S 1.09 121 S121L 1.35 121 S121W 1.33 121 S121R 1.26 121 S121K 1.24 121 S121G 1.20 121 S121C 1.18 121 S121N 1.14 121 S121T 1.13 121 S121A 1.12 121 S121V 1.12 122 A122H 1.14 122 A122I 1.13 122 A122T 1.08 122 A122K 1.08 122 A122V 1.04 122 A122S 1.03 123 G123D 1.73 123 G123V 1.40 123 G123P 1.32 123 G123E 1.13 123 G123T 1.06 123 G123H 1.00 124 G124L 1.92 124 G124I 1.85 124 G124T 1.64 124 G124H 1.59 124 G124V 1.44 124 G124F 1.32 124 G124S 1.27 124 G124Y 1.23 124 G124R 1.14 124 G124Q 1.12 125 V125G 2.95 125 V125S 1.94 125 V125A 1.69 125 V125P 1.50 125 V125R 1.30 125 V125D 1.24 125 V125Y 1.08 125 V125I 1.01 126 G126T 1.58 126 G126P 1.17 126 G126L 1.17 127 T127H 1.57 127 T127V 1.07 127 T127I 1.06 127 T127S 1.05 128 T128L 1.06 128 T128K 1.06 130 P130T 1.19 130 P130H 1.17 130 P130K 1.16 130 P130G 1.16 130 P130S 1.16 130 P130V 1.15 130 P130W 1.15 130 P130I 1.12 130 P130L 1.12 130 P130R 1.11 130 P130F 1.08 130 P130E 1.00 131 A131L 1.83 131 A131R 1.76 131 A131H 1.72 131 A131G 1.66 131 A131W 1.61 131 A131V 1.59 131 A131P 1.52 131 A131Y 1.50 131 A131S 1.48 131 A131E 1.36 131 A131D 1.31 131 A131Q 1.29 132 P132Y 1.57 132 P132S 1.13 133 K133Y 1.12 133 K133L 1.05 133 K133H 1.02 134 V134G 1.71 134 V134T 1.25 134 V134N 1.18 134 V134S 1.16 134 V134L 1.13 134 V134I 1.12 136 V136T 1.13 137 V137M 1.22 137 V137L 1.09 137 V137T 1.08 137 V137A 1.07 137 V137G 1.02 138 S138I 1.15 138 S138G 1.05 140 P140A 1.90 140 P140T 1.74 140 P140S 1.31 141 P141L 2.32 141 P141I 2.29 141 P141H 2.07 141 P141V 1.96 141 P141T 1.84 141 P141S 1.70 141 P141R 1.65 141 P141G 1.64 141 P141Q 1.39 141 P141N 1.32 141 P141A 1.10 142 L142W 2.41 142 L142K 1.60 142 L142F 1.05 143 A143K 3.16 143 A143H 2.90 143 A143L 2.51 143 A143V 2.45 143 A143W 2.27 143 A143T 2.18 143 A143R 2.15 143 A143S 1.77 143 A143Q 1.74 143 A143F 1.56 143 A143P 1.53 143 A143G 1.48 143 A143D 1.45 143 A143E 1.43 143 A143C 1.39 143 A143N 1.30 144 P144Y 2.34 144 P144K 2.09 144 P144H 1.94 144 P144F 1.82 144 P144R 1.76 144 P144S 1.69 144 P144T 1.46 144 P144G 1.45 144 P144D 1.45 144 P144N 1.44 144 P144L 1.43 144 P144Q 1.37 144 P144M 1.24 144 P144A 1.09 145 M145L 1.72 145 M145F 1.49 145 M145R 1.15 145 M145W 1.15 145 M145C 1.02 145 M145T 1.01 147 H147A 1.28 147 H147S 1.26 147 H147T 1.20 147 H147P 1.12 147 H147E 1.11 148 P148V 2.43 148 P148K 1.79 148 P148L 1.64 148 P148A 1.64 148 P148R 1.51 148 P148T 1.50 148 P148Y 1.46 148 P148S 1.46 148 P148E 1.42 148 P148F 1.37 148 P148Q 1.33 148 P148D 1.03 150 F150L 1.29 150 F150E 1.23 151 Q151D 1.47 151 Q151R 1.36 151 Q151P 1.35 151 Q151A 1.29 151 Q151T 1.24 151 Q151M 1.24 151 Q151E 1.14 151 Q151K 1.07 151 Q151H 1.06 151 Q151S 1.05 151 Q151C 1.05 151 Q151Y 1.01 152 L152V 1.22 152 L152K 1.21 152 L152R 1.20 152 L152W 1.18 152 L152T 1.12 152 L152S 1.12 152 L152Y 1.09 152 L152H 1.09 152 L152G 1.08 152 L152E 1.08 152 L152Q 1.07 152 L152D 1.07 152 L152I 1.04 152 L152C 1.00 153 I153K 1.62 153 I153H 1.46 153 I153T 1.27 153 I153L 1.27 153 I153F 1.23 153 I153A 1.19 154 F154Y 1.32 155 E155T 1.49 155 E155R 1.47 155 E155L 1.31 155 E155Y 1.27 155 E155K 1.23 155 E155G 1.17 155 E155S 1.08 155 E155D 1.08 155 E155F 1.07 156 G156P 1.44 156 G156T 1.15 156 G156K 1.10 156 G156M 1.09 156 G156C 1.07 156 G156N 1.07 156 G156R 1.05 156 G156H 1.04 156 G156S 1.02 157 G157T 1.74 157 G157R 1.51 157 G157S 1.30 157 G157K 1.28 157 G157F 1.27 157 G157V 1.23 157 G157H 1.14 157 G157I 1.11 158 E158H 2.40 158 E158K 2.08 158 E158F 2.06 158 E158R 1.99 158 E158Y 1.77 158 E158W 1.77 158 E158L 1.59 158 E158S 1.57 158 E158V 1.52 158 E158Q 1.49 158 E158C 1.46 158 E158A 1.45 158 E158T 1.45 158 E158P 1.41 158 E158N 1.41 158 E158M 1.39 158 E158I 1.38 158 E158D 1.35 159 Q159R 1.15 159 Q159C 1.13 159 Q159S 1.10 159 Q159D 1.09 159 Q159A 1.08 159 Q159M 1.07 159 Q159P 1.06 159 Q159L 1.02 161 T161R 3.61 161 T161Y 2.40 161 T161H 1.82 161 T161W 1.41 161 T161I 1.40 161 T161V 1.27 161 T161L 1.25 161 T161Q 1.04 162 T162K 1.22 162 T162R 1.17 162 T162W 1.15 162 T162Y 1.03 162 T162H 1.02 163 E163L 1.50 163 E163Y 1.41 163 E163H 1.32 163 E163G 1.25 163 E163W 1.21 163 E163V 1.13 163 E163R 1.12 163 E163S 1.12 163 E163A 1.11 163 E163C 1.11 163 E163F 1.07 165 A165R 1.70 165 A165K 1.35 165 A165F 1.23 165 A165Q 1.21 165 A165V 1.21 165 A165Y 1.20 165 A165T 1.18 165 A165I 1.17 165 A165P 1.14 165 A165L 1.08 165 A165G 1.05 165 A165N 1.01 165 A165S 1.00 166 R166Y 1.29 166 R166L 1.27 166 R166I 1.26 166 R166W 1.25 166 R166H 1.20 166 R166T 1.19 166 R166V 1.17 166 R166K 1.17 166 R166S 1.16 166 R166G 1.15 167 V167T 1.13 167 V167I 1.08 167 V167Y 1.07 167 V167H 1.03 168 Y168G 1.89 168 Y168T 1.51 168 Y168V 1.19 169 S169Y 1.26 169 S169R 1.24 169 S169K 1.21 169 S169I 1.16 169 S169T 1.15 169 S169L 1.08 169 S169C 1.03 169 S169Q 1.02 170 A170K 1.71 170 A170G 1.59 170 A170I 1.59 170 A170S 1.47 170 A170F 1.44 170 A170T 1.40 170 A170E 1.28 170 A170D 1.27 170 A170N 1.21 170 A170V 1.20 170 A170C 1.15 170 A170Q 1.15 170 A170L 1.05 170 A170W 1.04 170 A170M 1.03 171 L171K 2.05 171 L171H 1.67 171 L171T 1.54 171 L171I 1.53 171 L171S 1.43 171 L171F 1.30 171 L171G 1.26 171 L171Y 1.20 171 L171V 1.02 172 A172I 1.70 172 A172S 1.59 172 A172W 1.43 172 A172G 1.41 172 A172V 1.40 172 A172T 1.25 172 A172L 1.20 172 A172C 1.20 173 S173Y 1.19 173 S173K 1.17 173 S173W 1.16 173 S173L 1.15 173 S173R 1.09 173 S173H 1.07 173 S173T 1.06 174 F174G 1.60 174 F174P 1.54 174 F174Q 1.42 174 F174C 1.32 174 F174S 1.16 174 F174L 1.05 175 M175T 2.21 175 M175G 2.04 175 M175V 1.93 175 M175L 1.61 175 M175Q 1.56 175 M175R 1.55 175 M175N 1.39 175 M175W 1.25 176 K176W 1.19 176 K176T 1.04 176 K176Y 1.04 176 K176V 1.04 176 K176G 1.01 178 P178L 1.82 178 P178Y 1.38 178 P178K 1.34 178 P178W 1.14 178 P178G 1.09 179 F179L 1.15 179 F179Y 1.05 180 F180L 1.30 180 F180I 1.20 180 F180V 1.14 180 F180Y 1.12 180 F180W 1.11 180 F180K 1.08 180 F180T 1.01 181 D181A 1.35 181 D181K 1.33 181 D181Y 1.29 181 D181W 1.26 181 D181L 1.25 181 D181R 1.23 181 D181S 1.21 181 D181Q 1.14 181 D181E 1.10 181 D181G 1.09 181 D181C 1.09 181 D181P 1.03 181 D181T 1.02 182 A182T 1.14 184 S184Y 1.06 184 S184F 1.05 184 S184T 1.04 184 S184H 1.02 185 V185K 1.37 185 V185Y 1.37 185 V185W 1.36 185 V185H 1.30 185 V185L 1.23 185 V185R 1.15 185 V185G 1.12 185 V185T 1.11 185 V185S 1.09 185 V185I 1.07 185 V185F 1.02 186 I186G 1.86 186 I186T 1.51 186 I186A 1.46 186 I186S 1.39 186 I186V 1.28 186 I186L 1.17 186 I186F 1.01 187 S187K 1.45 187 S187Y 1.43 187 S187I 1.38 187 S187L 1.37 187 S187W 1.30 187 S187H 1.29 187 S187V 1.23 187 S187T 1.12 187 S187R 1.04 187 S187G 1.03 187 S187F 1.02 188 T188Y 1.48 188 T188V 1.22 188 T188S 1.16 188 T188I 1.13 188 T188H 1.11 188 T188R 1.01 189 D189L 1.30 189 D189H 1.25 189 D189W 1.09 190 G190W 1.88 190 G190K 1.01 191 V191Y 1.32 191 V191H 1.30 191 V191W 1.20 191 V191S 1.20 191 V191K 1.17 191 V191I 1.14 191 V191F 1.13 191 V191R 1.05 191 V191L 1.04 196 F196H 1.77 196 F196L 1.77 196 F196C 1.74 196 F196M 1.65 196 F196G 1.59 196 F196S 1.58 196 F196Y 1.41 196 F196V 1.40 196 F196I 1.32 196 F196W 1.01 197 T197L 1.21 198 E198R 1.82 198 E198I 1.80 198 E198V 1.60 198 E198W 1.59 198 E198L 1.57 198 E198P 1.52 198 E198Y 1.48 198 E198C 1.38 198 E198F 1.37 198 E198Q 1.28 198 E198T 1.25 198 E198N 1.24 198 E198M 1.18 198 E198S 1.06 199 A199C 1.77 199 A199K 1.72 199 A199E 1.56 199 A199L 1.38 199 A199T 1.33 199 A199R 1.33 199 A199V 1.32 199 A199D 1.31 199 A199H 1.27 199 A199Y 1.24 199 A199F 1.23 199 A199S 1.20 199 A199G 1.14 199 A199M 1.07 201 N201Y 1.29 201 N201F 1.16 201 N201G 1.08 202 R202W 1.97 202 R202F 1.89 202 R202E 1.69 202 R202H 1.64 202 R202T 1.55 202 R202S 1.49 202 R202A 1.48 202 R202C 1.44 202 R202M 1.43 202 R202L 1.43 202 R202G 1.39 202 R202I 1.33 203 D203L 2.42 203 D203R 2.23 203 D203I 1.99 203 D203W 1.99 203 D203F 1.92 203 D203H 1.84 203 D203C 1.78 203 D203S 1.66 203 D203V 1.66 203 D203G 1.63 203 D203Q 1.60 203 D203A 1.53 203 D203E 1.34 203 D203N 1.05 The following Table, provides variants with a PAF PI greater than 1.5.

TABLE 10-3 PAF PI > 1.5 Wild-Type Residue/Pos. Variant Amino Acid(s) A2 W C7 H, I, K, Y D10 K, L, W, Y L12 C, Q G15 A E20 C, G, H, L, S, T, V, W D21 K, W G22 A T25 G, S E26 A, M A29 G, R, V, W, Y D31 L, W Q40 G, I, K, L, N, R, S, T, W, Y L42 H, K, W A44 C, F, L, V E47 R, T L53 H S54 A, I, L, P, R, V A55 G, T T57 R, S P63 G P66 H, I, R, V R67 N N69 K, V A71 G, H, I, S, T L74 G, S P75 R L78 E, G, H, N, Q, T, V A79 H, I, L H81 K D85 H, N, Q, R, S T93 F, Y D95 E K97 R A98 G, H, I, L, S, T, Y P104 C, E, F, G, H, I, N, R, S, T, V, W L105 C, P I107 E, G, N, S L109 N M111 I, K, L V113 L Q117 A, F, G, H, M, R, S, T, V, W, Y G123 D, H, I, L, T G124 I, L V125 A, G, P, S G126 T T127 H A131 G, H, L, P, R, V, W, Y P132 Y V134 G P140 A, T, P141 G, H, I, L, R, S, T, V L142 K, W A143 F, H, K, L, P, Q, R, S, T, V, W P144 F, H, K, R, S, Y M145 L P148 A, K, L, R, T, V I153 K G157 R, T E158 F, H, K, L, R, S, V, W, Y T161 H, R, Y A165 T Y168 G, T A170 G, I, K L171 H, I, K, T A172 I, S F174 G, P M175 G, L, Q, R, T, V P178 L F196 C, G, H, L, M, S G190 W E198 I, L, P, R, V, W A199 C, E, K R202 E, F, H, T, W D203 A, C, F, G, H, I, L, Q, R,S, V, W V206 E, F, G, H, K, R, S, A209 K E210 H, K, S, T, V, W Q211 K V212 W

Table 10-4 provides variants with PAF PI values greater than 2.0.

TABLE 10-4 Variants with PAF PI > 2.0 Wild-Type Residue/Pos. Amino Acid Variant(s) C7 K, Y D10 K. L, W L12 C, Q E20 G, H, L, S, T, V, W E26 M Q40 I, K, L, T, W L42 K, W A44 F, V E47 R L53 H S54 A, I, L, P, R, V L74 S L78 E, G, H, N D85 Q, R, S T93 F, Y D95 E K97 R A98 H, I, L, Y P104 G, H, I, S, T. V, W I107 E, S Q117 A, G, H, T, V, W, Y V125 G P141 H, I, L L142 W A143 H, K, L, R, T, V, W P144 K, Y P148 V E158 F, H, K T161 R, Y L171 K M175 G, T D203 L, R V206 E, F, K E210 T

The following Table provides PAD assay results for various variants.

TABLE 10-5 PAD Assay Results WT/Pos/ PAD Perf. Position Mutation Variant Ind. 1 M001A A <0.01 1 M001E E <0.01 1 M001F F <0.01 1 M001G G <0.01 1 M001K K <0.01 1 M001N N <0.01 1 M001P P <0.01 1 M001R R <0.01 1 M001S S <0.01 1 M001T T <0.01 1 M001W W <0.01 1 M001V V 0.944944 3 K003V V 0.835476 4 R004L L <0.01 4 R004V V 0.079216 4 R004I I 0.153122 4 R004W W 0.484006 4 R004G G 0.78952 4 R004S S 0.907174 4 R004E E 0.970668 4 R004Y Y 0.983327 4 R004H H 0.986096 4 R004Q Q 0.98766 4 R004T T 0.999841 5 I005G G <0.01 5 I005N N <0.01 5 I005P P <0.01 5 I005R R <0.01 5 I005W W <0.01 5 I005F F 0.15045 5 I005S S 0.367738 5 I005H H 0.626022 5 I005T T 0.7212 5 I005V V 0.917243 6 L006S S <0.01 6 L006K K <0.01 6 L006G G <0.01 6 L006H H <0.01 6 L006R R <0.01 6 L006W W <0.01 6 L006E E <0.01 6 L006Q Q <0.01 6 L006V V 0.352616 6 L006T T 0.354148 6 L006I I 0.819654 7 C007S S <0.01 7 C007R R <0.01 7 C007L L <0.01 7 C007P P <0.01 7 C007T T <0.01 7 C007W W <0.01 7 C007Y Y 0.544454 7 C007M M 0.678238 7 C007G G 0.686018 10 D010W W <0.01 10 D010K K <0.01 10 D010Y Y <0.01 10 D010T T <0.01 10 D010I I <0.01 10 D010V V <0.01 10 D010S S <0.01 10 D010G G <0.01 10 D010R R <0.01 10 D010A A <0.01 10 D010M M <0.01 10 D010N N <0.01 10 D010P P <0.01 10 D010E E 0.147899 11 S011T T <0.01 11 S011V V <0.01 11 S011D D <0.01 11 S011E E <0.01 11 S011F F <0.01 11 S011G G <0.01 11 S011L L <0.01 11 S011Q Q <0.01 11 S011R R <0.01 11 S011H H 0.332012 11 S011K K 0.399168 11 S011A A 0.528328 11 S011I I 0.562735 12 L012V V <0.01 12 L012S S <0.01 12 L012G G <0.01 12 L012R R <0.01 12 L012D D <0.01 12 L012P P <0.01 12 L012W W <0.0162738575856614 12 L012T T 0.064264 12 L012A A 0.074567 12 L012K K 0.134919 12 L012H H 0.164894 12 L012F F 0.171369 12 L012Q Q 0.219754 12 L012C C 0.221492 12 L012N N 0.655242 13 T013F F <0.01 13 T013R R <0.01 13 T013W W <0.01 13 T013Q Q 0.508867 13 T013V V 0.625148 13 T013S S 0.682494 13 T013G G 0.768701 14 W014I I <0.01 14 W014S S <0.01 14 W014G G <0.01 14 W014K K <0.01 14 W014V V <0.01 14 W014L L <0.01 14 W014T T <0.01 14 W014R R <0.01 14 W014N N <0.01 14 W014P P <0.01 14 W014E E 0.150043 14 W014F F 0.218073 14 W014A A 0.271277 14 W014Y Y 0.64896 14 W014W W 0.989643 15 G015C C <0.01 15 G015N N <0.01 15 G015D D <0.01 15 G015E E <0.01 15 G015H H <0.01 15 G015K K <0.01 15 G015L L <0.01 15 G015P P <0.01 15 G015R R <0.01 15 G015Y Y <0.01 15 G015A A 0.614319 15 G015S S 0.631317 16 W016S S <0.01 16 W016G G <0.01 16 W016H H <0.01 16 W016N N <0.01 16 W016R R <0.01 16 W016T T <0.01 16 W016P P 0.150383 16 W016Q Q 0.312038 16 W016M M 0.370155 16 W016A A 0.553088 16 W016D D 0.569713 16 W016E E 0.647375 16 W016V V 0.875327 17 V017A A 0.675391 17 V017E E 0.749717 17 V017G G 0.838345 17 V017K K 0.844479 17 V017F F 0.847091 17 V017T T 0.861827 17 V017Y Y 0.876678 17 V017R R 0.936013 17 V017P P 0.956795 17 V017I I 0.993337 17 V017L L 0.996217 18 P018A A <0.01 18 P018M M <0.01 18 P018S S 0.066689 19 V019P P <0.01 19 V019M M 0.117174 19 V019R R 0.343385 19 V019Q Q 0.395965 19 V019A A 0.554598 19 V019G G 0.55596 19 V019S S 0.573928 19 V019E E 0.620236 19 V019Y Y 0.696626 19 V019D D 0.785756 19 V019L L 0.910961 19 V019K K 0.965611 21 D021V V <0.01 21 D021P P 0.534939 21 D021S S 0.689672 21 D021E E 0.864655 21 D021F F 0.876655 21 D021W W 0.894205 21 D021L L 0.971454 22 G022K K <0.01 22 G022W W 0.231005 22 G022R R 0.563069 22 G022V V 0.850851 22 G022S S 0.981692 23 A023R R 0.283095 23 A023S S 0.335177 23 A023G G 0.350575 23 A023F F 0.438047 23 A023V V 0.598414 23 A023Q Q 0.732052 23 A023P P 0.733451 23 A023W W 0.801206 23 A023M M 0.946802 23 A023Y Y 0.962455 24 P024S S 0.614708 24 P024Q Q 0.652848 24 P024T T 0.663925 24 P024A A 0.681992 24 P024G G 0.755229 24 P024I I 0.853247 24 P024R R 0.907892 24 P024H H 0.969695 25 T025P P <0.01 25 T025H H <0.01 25 T025L L <0.01 25 T025R R <0.01 25 T025M M <0.01 25 T025E E <0.01 25 T025D D <0.01 25 T025K K 0.133406 25 T025W W 0.144315 25 T025I I 0.350917 25 T025G G 0.426214 25 T025C C 0.509792 25 T025V V 0.514769 25 T025S S 0.576256 25 T025A A 0.863346 26 E026S S 0.280953 26 E026T T 0.39705 26 E026W W 0.471182 26 E026N N 0.47572 26 E026R R 0.813632 26 E026G G 0.869755 26 E026C C 0.939981 26 E026V V 0.966156 26 E026P P 0.993535 27 R027W W <0.01 27 R027T T <0.0149789677895526 27 R027P P 0.483512 27 R027C C 0.58498 27 R027S S 0.686775 27 R027G G 0.836174 27 R027E E 0.925988 27 R027V V 0.943209 28 F028G G <0.01 28 F028H H <0.01 28 F028I I <0.01 28 F028R R <0.01 28 F028P P 0.385272 28 F028V V 0.531941 28 F028S S 0.696363 29 A029V V 0.43718 29 A029T T 0.467508 29 A029S S 0.546873 29 A029Y Y 0.593264 29 A029P P 0.622623 29 A029R R 0.728312 29 A029W W 0.738583 29 A029M M 0.768108 29 A029G G 0.802278 29 A029E E 0.844095 29 A029D D 0.996225 30 P030M M 0.78893 30 P030Q Q 0.905135 30 P030A A 0.918048 31 D031E E 0.882779 32 V032P P <0.01 32 V032R R 0.715259 33 R033D D <0.01 33 R033E E <0.01 33 R033H H <0.01 33 R033P P <0.01 33 R033W W <0.01 33 R033V V 0.935183 34 W034R R <0.01 34 W034E E <0.01 34 W034K K <0.01 34 W034Q Q 0.041311 34 W034S S 0.079486 34 W034T T 0.153641 34 W034V V 0.72591 34 W034G G 0.880049 34 W034I I 0.93831 35 T035Q Q <0.01 35 T035N N <0.01 35 T035R R <0.01 35 T035K K <0.01 35 T035L L <0.01 35 T035P P <0.01 35 T035W W <0.01 35 T035Y Y <0.01 35 T035V V 0.344374 36 G036P P <0.01 36 G036S S 0.25722 36 G036T T 0.326076 36 G036V V 0.375828 36 G036M M 0.536338 36 G036N N 0.557724 36 G036W W 0.682701 36 G036Q Q 0.712029 36 G036R R 0.897684 38 L038K K <0.01 38 L038G G <0.01 38 L038E E <0.01 38 L038P P <0.01 38 L038Q Q <0.01 38 L038R R <0.01 38 L038W W <0.01 40 Q040P P <0.01 41 Q041V V <0.01 41 Q041S S 0.222419 41 Q041P P 0.662368 41 Q041Y Y 0.701492 41 Q041W W 0.878483 42 L042W W <0.01 42 L042H H <0.01 42 L042T T <0.01 42 L042D D <0.01 42 L042Q Q 0.280991 42 L042S S 0.450557 42 L042R R 0.64188 42 L042I I 0.658658 42 L042V V 0.725221 42 L042M M 0.73687 42 L042G G 0.759964 43 G043S S 0.233902 43 G043P P 0.310899 43 G043V V 0.332639 43 G043Q Q 0.475759 43 G043R R 0.585481 43 G043C C 0.725373 43 G043I I 0.766408 43 G043K K 0.856798 43 G043M M 0.877674 43 G043Y Y 0.944457 43 G043H H 0.957156 44 A044S S <0.01 44 A044Y Y <0.01 44 A044T T <0.01 44 A044R R <0.01 44 A044D D <0.01 44 A044H H <0.01 44 A044P P <0.01 44 A044E E 0.028463 44 A044V V 0.504951 44 A044F F 0.803847 44 A044W W 0.847767 44 A044M M 0.975188 44 A044L L 0.99381 45 D045S S 0.382964 45 D045T T 0.438291 45 D045R R 0.492492 45 D045V V 0.500129 45 D045P P 0.531241 45 D045Q Q 0.568687 45 D045W W 0.582004 45 D045H H 0.779564 45 D045L L 0.781626 45 D045M M 0.78286 45 D045G G 0.839279 45 D045A A 0.841569 45 D045C C 0.844725 45 D045K K 0.867296 46 F046H H <0.01 46 F046T T 0.429962 46 F046W W 0.633171 46 F046S S 0.656356 46 F046V V 0.786355 46 F046I I 0.882982 46 F046G G 0.944614 47 E047P P 0.357072 47 E047R R 0.620501 47 E047N N 0.627512 47 E047S S 0.628088 47 E047M M 0.703134 47 E047A A 0.757492 47 E047F F 0.763159 47 E047C C 0.772744 47 E047T T 0.837562 47 E047D D 0.975388 47 E047H H 0.99217 48 V048R R <0.01 48 V048W W <0.01 48 V048S S 0.423613 48 V048G G 0.873544 48 V048N N 0.980906 48 V048E E 0.987222 49 I049P P 0.161279 49 I049R R 0.29139 49 I049W W 0.676641 49 I049H H 0.740799 49 I049S S 0.789362 49 I049E E 0.876247 49 I049V V 0.972022 50 E050R R <0.01 50 E050W W 0.14091 50 E050V V 0.425221 50 E050I I 0.575369 50 E050S S 0.645021 50 E050Q Q 0.906441 50 E050L L 0.967983 51 E051R R <0.01 51 E051P P <0.01 51 E051I I 0.044391 51 E051W W 0.165053 51 E051V V 0.367755 51 E051Q Q 0.761883 51 E051L L 0.927544 52 G052H H <0.01 52 G052S S <0.01 52 G052V V <0.01 52 G052T T <0.01 52 G052M M <0.01 52 G052F F <0.01 52 G052I I 0.069022 52 G052P P 0.242545 52 G052L L 0.244397 52 G052Q Q 0.283827 52 G052R R 0.349923 52 G052E E 0.549067 52 G052A A 0.793929 53 L053R R <0.01 53 L053W W <0.01 53 L053P P <0.01 53 L053D D <0.01328259968325 53 L053E E 0.191623 53 L053K K 0.237686 53 L053S S 0.260431 53 L053G G 0.32712 53 L053V V 0.652864 53 L053I I 0.659806 53 L053Q Q 0.717093 53 L053T T 0.842042 54 S054F F <0.01 54 S054W W <0.01 54 S054H H <0.01 54 S054K K 0.083519 54 S054I I 0.116295 54 S054Y Y 0.124722 54 S054G G 0.170484 54 S054L L 0.258821 54 S054V V 0.285755 54 S054E E 0.296919 54 S054T T 0.329279 54 S054R R 0.354857 54 S054M M 0.482666 54 S054Q Q 0.531633 54 S054D D 0.647787 54 S054C C 0.87772 55 A055V V <0.01 55 A055I I <0.01 55 A055P P <0.01 55 A055W W <0.01 55 A055Y Y 0.176777 55 A055R R 0.245648 55 A055T T 0.415054 55 A055G G 0.731513 55 A055L L 0.866592 55 A055S S 0.866756 55 A055H H 0.921909 56 R056C C <0.01 56 R056G G <0.01 56 R056T T <0.01 56 R056E E <0.01 56 R056H H <0.01 56 R056K K <0.01 56 R056P P <0.01 56 R056Q Q <0.01 56 R056W W <0.01 56 R056Y Y <0.01 56 R056S S 0.123501 56 R056L L 0.237933 56 R056N N 0.267811 56 R056A A 0.68802 57 T057R R <0.01 57 T057P P <0.01 57 T057W W <0.01 57 T057N N 0.245605 57 T057C C 0.398001 57 T057Y Y 0.551709 57 T057H H 0.605386 57 T057A A 0.651879 57 T057L L 0.762087 57 T057V V 0.86913 57 T057I I 0.870692 58 T058E E <0.01 58 T058G G <0.01 58 T058K K <0.01 58 T058P P <0.01 58 T058R R <0.01 58 T058W W <0.01 58 T058Y Y <0.01 58 T058M M 0.026886 58 T058A A 0.361258 58 T058V V 0.955494 58 T058S S 0.964758 59 N059R R <0.01 59 N059M M <0.01 59 N059P P <0.01 59 N059Q Q 0.165409 59 N059T T 0.501362 59 N059S S 0.651989 59 N059K K 0.731191 59 N059E E 0.879272 59 N059V V 0.887341 59 N059G G 0.890006 59 N059F F 0.911279 59 N059A A 0.929578 59 N059Y Y 0.99189 59 N059C C 0.99959 60 I060P P 0.318965 60 I060D D 0.660273 60 I060C C 0.668516 60 I060M M 0.682237 60 I060A A 0.788799 60 I060R R 0.809655 60 I060L L 0.913226 60 I060E E 0.923286 60 I060K K 0.959958 60 I060S S 0.999829 61 D061F F 0.698154 61 D061A A 0.708121 61 D061C C 0.848446 61 D061Y Y 0.948278 61 D061V V 0.968066 61 D061N N 0.999276 62 D062T T <0.01 62 D062I I <0.01 62 D062V V <0.01 62 D062H H <0.01 62 D062W W <0.01 62 D062S S <0.01 62 D062L L <0.01 62 D062G G <0.01 62 D062R R <0.01 62 D062M M <0.01 62 D062P P <0.01 62 D062Q Q <0.01 62 D062A A 0.113753 62 D062C C 0.490736 62 D062E E 0.602369 63 P063A A 0.598416 63 P063R R 0.801911 63 P063S S 0.898408 63 P063M M 0.908904 63 P063F F 0.925844 63 P063Y Y 0.948378 64 T064R R 0.106209 64 T064D D 0.640095 64 T064W W 0.691185 64 T064Q Q 0.865168 64 T064C C 0.876862 64 T064P P 0.936023 64 T064H H 0.960718 64 T064N N 0.983933 64 T064S S 0.987972 65 D065V V 0.199467 65 D065R R 0.215599 65 D065H H 0.398178 65 D065Y Y 0.42301 65 D065P P 0.423122 65 D065S S 0.468174 65 D065W W 0.50219 65 D065T T 0.5039 65 D065G G 0.51655 65 D065I I 0.617391 65 D065A A 0.723321 66 P066N N 0.381273 66 P066Q Q 0.422614 66 P066G G 0.444859 66 P066R R 0.508806 66 P066C C 0.523524 66 P066A A 0.563865 66 P066F F 0.672865 66 P066Y Y 0.699931 66 P066D D 0.718749 66 P066I I 0.844376 66 P066V V 0.89302 66 P066H H 0.947771 66 P066L L 0.987271 67 R067F F <0.0149736260903786 67 R067W W <0.0171329732205367 67 R067P P 0.036575 67 R067E E 0.113415 67 R067V V 0.1203 67 R067Q Q 0.126838 67 R067L L 0.156654 67 R067A A 0.215271 67 R067T T 0.315404 67 R067N N 0.333066 67 R067G G 0.40823 67 R067K K 0.986487 68 L068G G <0.01 68 L068A A <0.01 68 L068M M 0.02834 68 L068C C 0.05996 68 L068S S 0.071622 68 L068N N 0.100981 68 L068E E 0.131505 68 L068H H 0.222734 68 L068Q Q 0.254448 68 L068F F 0.254797 68 L068T T 0.324904 68 L068P P 0.35297 68 L068D D 0.443469 68 L068Y Y 0.447862 68 L068R R 0.465293 68 L068V V 0.507389 68 L068W W 0.561612 68 L068I I 0.727312 69 N069Y Y 0.173925 69 N069W W 0.55063 69 N069P P 0.591783 69 N069R R 0.828172 69 N069G G 0.976332 70 G070M M <0.01 70 G070T T <0.01 70 G070P P <0.01 70 G070V V <0.01 70 G070C C <0.01 70 G070R R <0.01 70 G070Y Y <0.01 70 G070K K <0.01 70 G070N N <0.01 70 G070Q Q <0.01 70 G070F F <0.01 70 G070I I 0.270463 70 G070E E 0.33356 70 G070S S 0.638917 71 A071P P <0.01 71 A071N N 0.613838 71 A071D D 0.646588 71 A071G G 0.675895 71 A071S S 0.693249 71 A071R R 0.771492 71 A071H H 0.781953 71 A071I I 0.786894 71 A071T T 0.79386 71 A071E E 0.809505 71 A071L L 0.838126 71 A071F F 0.985677 71 A071C C 0.993683 72 S072Y Y 0.069096 72 S072W W 0.339835 72 S072P P 0.555612 72 S072Q Q 0.655328 72 S072L L 0.703483 72 S072R R 0.742354 72 S072D D 0.800127 72 S072V V 0.82827 72 S072E E 0.930527 72 S072T T 0.973836 73 Y073P P <0.01 73 Y073R R 0.262561 73 Y073L L 0.497588 73 Y073G G 0.509699 73 Y073H H 0.515737 73 Y073I I 0.641914 73 Y073S S 0.676285 73 Y073V V 0.73535 73 Y073N N 0.758401 73 Y073D D 0.803442 73 Y073Q Q 0.866092 73 Y073K K 0.944166 76 S076W W <0.01 76 S076Y Y 0.177113 76 S076F F 0.461095 76 S076Q Q 0.900789 77 C077Y Y <0.01 77 C077R R <0.01 77 C077W W <0.01 77 C077F F <0.01 77 C077N N <0.01 77 C077P P <0.01 77 C077G G 0.181068 77 C077L L 0.734708 77 C077S S 0.764136 77 C077V V 0.802259 77 C077A A 0.912937 78 L078E E <0.01 78 L078N N <0.01 78 L078A A <0.01 78 L078P P <0.01 78 L078R R <0.01 78 L078S S <0.01 78 L078M M 0.477538 78 L078Q Q 0.519566 78 L078C C 0.779536 78 L078Y Y 0.809511 78 L078V V 0.827484 79 A079H H <0.01 79 A079F F <0.01 79 A079V V <0.01 79 A079C C 0.026887 79 A079Q Q 0.268704 79 A079E E 0.272158 79 A079N N 0.281684 79 A079M M 0.284387 79 A079R R 0.321618 79 A079W W 0.530746 79 A079T T 0.598368 79 A079I I 0.673986 79 A079S S 0.779628 79 A079G G 0.915372 79 A079P P 0.94147 79 A079L L 0.958677 80 T080W W <0.01 80 T080L L <0.01 80 T080K K <0.01 80 T080R R <0.01 80 T080E E <0.01 80 T080P P <0.01 80 T080H H 0.049717 80 T080Y Y 0.107973 80 T080I I 0.146188 80 T080N N 0.529867 82 L082R R <0.01 82 L082S S <0.01 82 L082W W <0.01 82 L082V V 0.187819 82 L082G G 0.310823 82 L082T T 0.377413 82 L082H H 0.468806 82 L082I I 0.508005 82 L082K K 0.508537 82 L082P P 0.516154 82 L082A A 0.976228 83 P083T T <0.01 83 P083V V 0.186837 83 P083L L 0.211018 83 P083H H 0.611439 83 P083W W 0.621496 83 P083G G 0.677444 83 P083S S 0.789585 83 P083Q Q 0.818267 83 P083D D 0.831344 83 P083F F 0.99445 84 L084W W <0.01 84 L084V V 0.416576 84 L084P P 0.43025 84 L084T T 0.438956 84 L084A A 0.453182 84 L084Q Q 0.516002 84 L084S S 0.550862 84 L084R R 0.565943 84 L084N N 0.665228 84 L084K K 0.79008 84 L084D D 0.85276 84 L084I I 0.870124 84 L084H H 0.993217 85 D085I I 0.100248 85 D085L L 0.241561 85 D085V V 0.25268 85 D085W W 0.341677 85 D085P P 0.543807 85 D085Y Y 0.554364 85 D085S S 0.675803 85 D085T T 0.708548 85 D085N N 0.781957 85 D085Q Q 0.988545 86 L086H H <0.01 86 L086S S <0.01 86 L086R R <0.01 86 L086E E <0.01 86 L086F F <0.01 86 L086Q Q <0.01 86 L086W W 0.077717 86 L086V V 0.120133 86 L086T T 0.284184 86 L086G G 0.696393 86 L086Y Y 0.815121 86 L086P P 0.987233 87 V087S S <0.01 87 V087G G <0.01 87 V087Y Y <0.01 87 V087R R <0.01 87 V087K K <0.01 87 V087D D <0.01 87 V087F F 0.103908 87 V087T T 0.147618 87 V087A A 0.16806 87 V087M M 0.751854 89 I089H H <0.01 89 I089S S <0.01 89 I089G G <0.01 89 I089W W <0.01 89 I089Q Q <0.01 89 I089D D <0.01 89 I089E E <0.01 89 I089R R <0.01 89 I089F F 0.745747 89 I089V V 0.820031 89 I089T T 0.900425 94 N094L L <0.01 94 N094T T <0.01 94 N094V V <0.01 94 N094H H <0.01 94 N094R R <0.01 94 N094W W <0.01 94 N094M M 0.031458 94 N094C C 0.072751 94 N094Y Y 0.123924 94 N094G G 0.532837 94 N094A A 0.74316 94 N094P P 0.789771 94 N094S S 0.877698 95 D095A A <0.01 95 D095C C <0.01 95 D095G G <0.01 95 D095H H <0.01 95 D095K K <0.01 95 D095L L <0.01 95 D095N N <0.01 95 D095Q Q <0.01 95 D095R R <0.01 95 D095S S <0.01 95 D095T T <0.01 95 D095V V <0.01 95 D095W W <0.01 95 D095Y Y <0.01 95 D095E E 0.754335 96 T096I I <0.01 96 T096W W <0.01 96 T096Y Y <0.01 96 T096R R 0.136108 96 T096V V 0.58611 96 T096S S 0.786547 96 T096P P 0.885134 97 K097Q Q <0.01 97 K097G G <0.01 97 K097I I <0.01 97 K097W W <0.01 97 K097L L <0.01 97 K097V V <0.01 97 K097Y Y <0.01 97 K097S S <0.01 97 K097T T <0.01 97 K097D D <0.01 97 K097M M 0.216645 97 K097A A 0.227977 97 K097P P 0.26585 97 K097R R 0.587184 99 Y099R R 0.291941 99 Y099V V 0.311502 99 Y099S S 0.367181 99 Y099W W 0.566038 99 Y099H H 0.591623 99 Y099I I 0.60574 99 Y099G G 0.700083 99 Y099P P 0.813989 99 Y099A A 0.822549 99 Y099L L 0.856204 100 F100W W <0.01 100 F100K K <0.01 100 F100D D <0.01 100 F100E E 0.152427 100 F100S S 0.852784 101 R101W W <0.01 101 R101K K 0.068708 101 R101Q Q 0.107171 101 R101V V 0.442582 101 R101D D 0.800722 101 R101Y Y 0.803109 101 R101P P 0.855496 101 R101N N 0.918012 101 R101C C 0.946306 101 R101I I 0.955711 101 R101F F 0.965422 102 R102W W <0.01 102 R102F F 0.226881 102 R102G G 0.270733 102 R102C C 0.363718 102 R102V V 0.60605 102 R102D D 0.684234 102 R102P P 0.894709 102 R102S S 0.960127 103 T103W W <0.01 103 T103Y Y <0.01 103 T103G G <0.01 103 T103K K <0.01 103 T103I I <0.01 103 T103L L <0.01 103 T103H H <0.01 103 T103A A <0.01 103 T103V V <0.01 103 T103S S <0.01 103 T103C C <0.01 103 T103R R <0.01 103 T103N N <0.01 103 T103F F <0.01 103 T103P P <0.01 104 P104R R <0.01 104 P104A A <0.01 104 P104L L <0.01 104 P104W W 0.232802 104 P104T T 0.333526 104 P104S S 0.529113 104 P104Q Q 0.847699 104 P104F F 0.863543 104 P104G G 0.984538 105 L105V V <0.01 105 L105A A <0.01 105 L105M M <0.01 105 L105E E 0.528458 105 L105S S 0.609931 105 L105Y Y 0.620029 105 L105T T 0.638962 105 L105P P 0.902642 106 D106R R 0.559786 106 D106Q Q 0.617485 106 D106P P 0.632087 106 D106N N 0.642667 106 D106M M 0.855673 106 D106I I 0.915931 106 D106L L 0.99561 107 I107E E <0.01 107 I107G G <0.01 107 I107F F <0.01 107 I107Q Q <0.01 107 I107R R <0.01 107 I107H H <0.01 107 I107W W <0.01 107 I107P P 0.318743 107 I107Y Y 0.524182 107 I107A A 0.795478 107 I107N N 0.929935 107 I107V V 0.96863 108 A108D D <0.01 108 A108F F <0.01 108 A108H H <0.01 108 A108I I <0.01 108 A108N N <0.01 108 A108P P <0.01 108 A108R R <0.01 108 A108E E 0.60726 108 A108Q Q 0.734472 108 A108T T 0.865471 108 A108V V 0.950481 109 L109W W <0.01 109 L109D D 0.106206 109 L109I I 0.144257 109 L109E E 0.194168 109 L109R R 0.210346 109 L109H H 0.220153 109 L109Q Q 0.222755 109 L109F F 0.317718 109 L109A A 0.323528 109 L109S S 0.378623 109 L109P P 0.434661 109 L109G G 0.51022 109 L109V V 0.539733 109 L109M M 0.628881 109 L109N N 0.658369 109 L109T T 0.79132 109 L109Y Y 0.825105 110 G110T T <0.01 110 G110L L <0.01 110 G110W W <0.01 110 G110Y Y <0.01 110 G110P P 0.224284 110 G110I I 0.232219 110 G110S S 0.30218 110 G110Q Q 0.343918 110 G110R R 0.476072 110 G110H H 0.73456 110 G110N N 0.770851 110 G110M M 0.816422 111 M111R R <0.01 111 M111S S 0.139078 111 M111H H 0.192733 111 M111G G 0.315165 111 M111P P 0.566892 111 M111E E 0.668985 111 M111L L 0.67115 111 M111K K 0.706165 111 M111T T 0.763332 111 M111F F 0.776934 111 M111D D 0.78777 111 M111V V 0.92522 112 S112Y Y <0.01 112 S112R R <0.01 112 S112P P <0.01 112 S112H H 0.380254 112 S112V V 0.479716 112 S112M M 0.564157 112 S112W W 0.582165 112 S112K K 0.678369 112 S112T T 0.721644 112 S112N N 0.850159 112 S112F F 0.878895 112 S112A A 0.943049 113 V113S S 0.572415 113 V113G G 0.579385 113 V113K K 0.716865 113 V113H H 0.763416 113 V113W W 0.803685 113 V113L L 0.854963 113 V113T T 0.861744 113 V113D D 0.871104 113 V113E E 0.936465 113 V113C C 0.937598 113 V113F F 0.959822 113 V113Y Y 0.981976 114 L114H H <0.01 114 L114E E <0.01 114 L114F F <0.01 114 L114K K <0.01 114 L114R R <0.01 114 L114W W <0.01 114 L114Y Y <0.01 114 L114Q Q 0.115737 114 L114P P 0.275464 114 L114S S 0.545726 114 L114V V 0.595416 114 L114N N 0.77333 115 V115H H <0.01 115 V115K K <0.01 115 V115I I 0.994833 116 T116Y Y 0.466112 116 T116V V 0.571817 116 T116R R 0.619823 116 T116L L 0.681201 116 T116W W 0.748358 116 T116I I 0.760474 116 T116Q Q 0.768867 116 T116P P 0.836786 116 T116G G 0.901886 116 T116E E 0.906124 116 T116A A 0.952003 116 T116S S 0.963005 117 Q117W W 0.707035 117 Q117V V 0.761971 117 Q117G G 0.794858 117 Q117S S 0.86512 118 V118K K <0.01 118 V118W W <0.01 118 V118E E <0.01 118 V118R R 0.069623 118 V118P P 0.222399 118 V118D D 0.40168 118 V118I I 0.545694 118 V118G G 0.559239 118 V118S S 0.815888 118 V118A A 0.852723 118 V118T T 0.91759 118 V118M M 0.933469 118 V118F F 0.998467 119 L119G G <0.01 119 L119S S <0.01 119 L119F F <0.01 119 L119R R <0.01 119 L119P P <0.01 119 L119T T 0.102922 119 L119N N 0.113151 119 L119V V 0.150373 119 L119W W 0.203313 119 L119C C 0.244106 119 L119D D 0.280381 119 L119E E 0.322167 119 L119I I 0.427476 119 L119H H 0.462912 119 L119Y Y 0.556343 120 T120P P <0.01 120 T120H H 0.498304 120 T120R R 0.599376 120 T120A A 0.663543 120 T120Q Q 0.781096 120 T120C C 0.924433 121 S121P P 0.384623 121 S121R R 0.701237 121 S121W W 0.772781 121 S121K K 0.77795 121 S121G G 0.992545 122 A122G G <0.01 122 A122D D 0.059137 122 A122F F 0.148369 122 A122H H 0.169443 122 A122R R 0.396041 122 A122S S 0.431258 122 A122K K 0.450105 122 A122E E 0.467766 122 A122T T 0.520454 122 A122P P 0.548155 122 A122I I 0.647406 122 A122N N 0.704284 122 A122Q Q 0.741587 122 A122W W 0.862265 122 A122V V 0.886387 122 A122M M 0.938855 124 G124I I <0.01 124 G124H H <0.01 124 G124M M <0.01 124 G124W W <0.01 124 G124P P <0.01 124 G124A A 0.031196 124 G124Q Q 0.208313 124 G124T T 0.315233 124 G124V V 0.329769 124 G124R R 0.409769 124 G124L L 0.536625 124 G124S S 0.555215 124 G124Y Y 0.559199 124 G124N N 0.599171 124 G124D D 0.63784 124 G124C C 0.672179 124 G124F F 0.950801 125 V125W W 0.24527 125 V125E E 0.385171 125 V125R R 0.466062 125 V125C C 0.541228 125 V125D D 0.541318 125 V125P P 0.622352 125 V125F F 0.627367 125 V125S S 0.790998 125 V125Y Y 0.813593 125 V125A A 0.925641 125 V125I I 0.941326 126 G126I I <0.010426347441542 126 G126V V 0.175001 126 G126Y Y 0.234673 126 G126L L 0.540613 126 G126A A 0.552538 126 G126E E 0.599533 126 G126P P 0.673809 126 G126T T 0.737666 126 G126R R 0.761417 126 G126N N 0.846727 126 G126S S 0.902662 126 G126C C 0.980807 127 T127L L <0.01 127 T127E E <0.01 127 T127Q Q 0.151533 127 T127I I 0.203586 127 T127H H 0.60105 127 T127D D 0.61747 127 T127M M 0.639504 127 T127C C 0.653314 127 T127V V 0.683337 127 T127G G 0.710564 127 T127P P 0.773291 127 T127S S 0.828003 128 T128D D 0.662836 129 Y129W W <0.01 129 Y129G G <0.01 129 Y129K K <0.01 129 Y129V V <0.01 129 Y129T T 0.138769 129 Y129A A 0.173554 129 Y129R R 0.178362 129 Y129M M 0.211662 129 Y129D D 0.228506 129 Y129L L 0.270643 129 Y129N N 0.530034 129 Y129P P 0.588917 129 Y129C C 0.610384 129 Y129S S 0.692051 129 Y129F F 0.713199 146 P146W W 0.680806 146 P146T T 0.756105 146 P146V V 0.768041 146 P146S S 0.956673 148 P148Q Q 0.975963 149 W149R R <0.01 149 W149E E <0.01 149 W149P P <0.01 149 W149C C 0.1164 149 W149I I 0.235936 149 W149A A 0.311848 149 W149S S 0.329233 149 W149Q Q 0.402387 149 W149T T 0.440303 149 W149G G 0.44856 149 W149M M 0.494615 149 W149F F 0.495779 149 W149L L 0.637667 149 W149Y Y 0.747652 150 F150P P 0.31768 150 F150N N 0.362798 150 F150G G 0.458431 150 F150V V 0.511676 150 F150A A 0.539571 150 F150T T 0.580879 150 F150W W 0.622886 150 F150M M 0.625886 150 F150E E 0.727755 150 F150C C 0.778063 150 F150I I 0.78431 150 F150K K 0.848249 153 I153N N 0.890296 154 F154T T <0.01 154 F154D D <0.01 154 F154E E <0.01 154 F154G G <0.01 154 F154L L <0.01 154 F154P P <0.01 154 F154V V <0.01 154 F154S S 0.287767 154 F154Q Q 0.973299 194 I194S S <0.01 194 I194A A <0.01 194 I194C C <0.01 194 I194P P <0.01 194 I194F F <0.01 194 I194W W <0.01 194 I194R R <0.01 194 I194Y Y <0.01 194 I194G G 0.044503 194 I194L L 0.577811 194 I194V V 0.780569 196 F196H H <0.01 196 F196G G <0.01 196 F196S S <0.01 196 F196Q Q <0.01 196 F196A A <0.01 196 F196K K <0.01 196 F196N N <0.01 196 F196R R <0.01 196 F196W W 0.38122 196 F196P P 0.385754 196 F196V V 0.675769 196 F196M M 0.709899 196 F196Y Y 0.970105

The following Table provides variants that are better than wild-type at degrading peracids (i.e., the performance index for the variant is better than the wild-type).

TABLE 10-6 Variants with Peracid Degradation Greater Than Wild-Type Pos. WT/Pos./Var. PAD PI 1 M001I 1.19 1 M001L 2.11 2 A002D 1.05 2 A002R 1.17 2 A002W 1.17 2 A002P 1.17 2 A002Q 1.29 2 A002E 1.38 3 K003T 1.03 3 K003S 1.17 3 K003Q 1.19 3 K003R 1.29 3 K003Y 1.39 3 K003M 1.44 3 K003P 1.45 3 K003C 1.52 3 K003L 1.84 3 K003H 1.89 3 K003A 2.14 3 K003I 2.44 3 K003E 3.51 3 K003G 3.74 4 R004D 1.18 4 R004C 1.34 4 R004P 1.44 4 R004A 1.64 5 I005M 1.09 5 I005E 1.59 5 I005L 1.63 5 I005A 1.88 5 I005C 2.47 5 I005D 3.11 6 L006C 1.22 6 L006M 1.44 6 L006A 1.99 7 C007A 1.03 7 C007H 1.37 7 C007I 1.48 7 C007E 1.63 7 C007K 2.95 8 F008M 1.11 8 F008L 1.31 8 F008A 1.33 8 F008C 4.01 10 D010L 2.04 13 T013I 1.05 13 T013E 1.09 13 T013L 1.47 13 T013M 1.47 13 T013C 1.55 13 T013A 1.88 13 T013N 2.61 13 T013P 2.73 16 W016K 1.03 16 W016I 1.06 16 W016Y 1.09 16 W016L 1.16 17 V017S 1.04 18 P018N 1.42 18 P018Q 3.26 18 P018R 3.97 18 P018C 4.16 18 P018Y 4.17 18 P018V 4.85 18 P018E 4.87 18 P018G 4.96 18 P018H 6.05 18 P018L 7.40 20 E020D 1.14 20 E020S 1.18 20 E020H 1.20 20 E020T 1.25 20 E020V 1.27 20 E020A 1.28 20 E020W 1.30 20 E020N 1.34 20 E020P 1.43 20 E020Q 1.56 20 E020C 1.76 21 D021S 1.11 21 D021E 1.39 21 D021F 1.41 21 D021W 1.44 21 D021L 1.57 21 D021A 1.75 21 D021G 1.76 21 D021K 1.80 21 D021Y 2.01 22 G022I 1.03 22 G022T 1.16 22 G022E 1.19 22 G022L 1.35 22 G022P 1.36 22 G022Q 1.44 22 G022A 1.66 23 A023H 1.04 23 A023L 1.30 24 P024C 1.04 24 P024K 1.36 24 P024L 1.51 26 E026M 1.10 26 E026H 1.19 26 E026D 1.39 26 E026A 1.45 26 E026K 1.47 26 E026L 1.71 27 R027I 1.41 27 R027K 1.55 27 R027L 2.60 27 R027A 2.78 28 F028E 1.04 28 F028W 1.17 28 F028C 1.21 28 F028Y 1.36 28 F028M 1.37 28 F028A 1.48 28 F028L 2.02 28 F028D 2.07 29 A029C 1.15 30 P030H 1.08 30 P030G 1.09 30 P030R 1.14 30 P030L 1.17 30 P030E 1.24 30 P030Y 1.31 30 P030I 1.38 30 P030K 1.39 30 P030S 1.49 30 P030T 1.64 30 P030V 1.74 31 D031V 1.08 31 D031T 1.11 31 D031Q 1.13 31 D031W 1.14 31 D031G 1.16 31 D031A 1.18 31 D031S 1.23 31 D031F 1.39 31 D031R 1.49 31 D031N 1.55 31 D031L 1.61 32 V032S 1.09 32 V032N 1.61 32 V032W 1.71 32 V032Q 1.74 32 V032G 2.65 32 V032M 3.41 32 V032I 3.51 32 V032A 3.64 32 V032E 3.92 32 V032D 4.19 32 V032L 4.72 32 V032K 4.73 33 R033S 1.01 33 R033N 1.30 33 R033A 1.32 33 R033C 1.73 33 R033G 2.63 33 R033K 2.72 33 R033L 2.90 34 W034P 1.21 34 W034M 1.22 34 W034C 1.49 34 W034A 2.29 35 T035M 2.72 35 T035A 3.85 35 T035C 4.72 35 T035I 5.38 35 T035E 5.73 36 G036C 1.06 36 G036A 1.07 36 G036H 1.10 36 G036K 1.71 36 G036I 1.81 36 G036L 2.49 36 G036D 2.50 37 V037I 1.04 37 V037L 1.16 37 V037S 1.49 37 V037N 1.52 37 V037C 1.63 37 V037A 2.00 37 V037P 2.10 38 L038V 1.12 39 A039W 1.02 39 A039Y 1.13 40 Q040N 1.00 40 Q040I 1.10 40 Q040E 1.28 40 Q040R 1.48 40 Q040L 1.49 40 Q040D 1.59 40 Q040S 1.65 40 Q040T 1.81 40 Q040Y 2.02 40 Q040G 2.17 40 Q040W 2.59 40 Q040K 3.64 41 Q041G 1.09 41 Q041H 1.14 41 Q041R 1.27 41 Q041K 1.61 41 Q041L 1.92 41 Q041A 2.58 42 L042F 1.02 42 L042P 1.34 42 L042K 1.41 42 L042C 1.43 43 G043A 1.07 43 G043L 1.82 43 G043E 1.88 44 A044C 1.92 45 D045F 1.04 46 F046C 1.16 46 F046A 1.25 46 F046E 1.31 46 F046D 1.39 46 F046M 1.42 46 F046K 1.46 46 F046P 1.50 46 F046L 1.54 47 E047L 1.02 47 E047K 1.06 47 E047G 1.10 47 E047I 1.15 48 V048Q 1.39 48 V048F 1.42 48 V048A 1.63 48 V048M 1.79 48 V048C 2.25 48 V048L 2.29 48 V048P 3.08 49 I049Y 1.02 49 I049M 1.02 49 I049L 1.03 49 I049G 1.12 49 I049K 1.26 49 I049A 1.87 50 E050P 1.02 50 E050M 1.04 50 E050G 1.11 50 E050D 1.22 50 E050A 1.23 51 E051T 1.17 51 E051M 1.20 51 E051D 1.28 51 E051G 1.34 51 E051K 2.00 51 E051A 2.72 52 G052W 2.47 53 L053H 1.70 54 S054N 1.29 54 S054P 1.30 54 S054A 1.41 55 A055N 1.05 55 A055K 1.08 55 A055C 1.26 57 T057S 1.01 57 T057G 1.05 58 T058L 1.12 58 T058H 1.49 59 N059Q 1.86 59 N059T 5.63 59 N059S 7.32 59 N059K 8.21 59 N059E 9.88 59 N059V 9.97 59 N059G 10.00 59 N059F 10.23 59 N059A 10.44 59 N059Y 11.14 59 N059C 11.23 59 N059D 11.72 59 N059W 12.80 59 N059L 14.74 60 I060G 1.04 60 I060V 1.06 60 I060H 1.07 60 I060Y 1.19 61 D061P 1.13 61 D061Q 1.16 61 D061L 1.20 61 D061G 1.25 61 D061S 1.35 61 D061R 1.59 61 D061I 1.66 61 D061H 1.67 61 D061K 1.72 63 P063K 1.02 63 P063V 1.04 63 P063Q 1.05 63 P063W 1.11 63 P063G 1.22 63 P063L 1.23 63 P063T 1.32 64 T064G 1.08 64 T064M 1.09 64 T064A 1.20 64 T064L 1.22 66 P066S 1.02 66 P066T 1.10 69 N069D 1.11 69 N069A 1.13 69 N069Q 1.14 69 N069C 1.20 69 N069L 1.20 69 N069S 1.42 69 N069T 1.43 69 N069H 1.52 69 N069K 1.59 69 N069V 1.73 69 N069I 1.75 70 G070L 1.01 70 G070A 1.41 70 G070H 1.90 71 A071K 1.01 71 A071M 1.11 72 S072F 1.15 72 S072G 1.76 72 S072M 2.13 72 S072C 2.18 72 S072H 2.48 72 S072N 2.85 72 S072A 3.52 73 Y073M 1.13 73 Y073C 1.20 73 Y073A 1.40 74 L074F 1.13 74 L074M 1.21 74 L074A 2.90 75 P075E 1.19 75 P075L 1.19 75 P075W 1.31 75 P075Y 1.32 75 P075V 1.39 75 P075C 1.42 75 P075D 2.09 76 S076C 1.06 76 S076T 1.11 76 S076A 1.11 76 S076H 1.11 76 S076P 1.20 76 S076V 1.35 76 S076K 1.53 76 S076M 1.61 76 S076D 1.94 76 S076E 2.09 76 S076G 2.15 76 S076L 4.70 77 C077T 1.03 77 C077D 1.05 78 L078T 1.10 78 L078I 1.11 78 L078G 1.38 78 L078H 1.57 80 T080V 1.01 80 T080Q 1.07 80 T080A 1.11 80 T080C 1.15 80 T080S 1.40 80 T080G 1.50 81 H081N 1.00 81 H081L 1.03 81 H081W 1.09 81 H081C 1.09 81 H081A 1.45 81 H081M 1.54 82 L082M 1.06 83 P083C 1.01 83 P083R 1.09 83 P083N 1.10 83 P083K 1.16 83 P083E 1.26 83 P083M 1.88 83 P083A 2.36 84 L084F 1.01 84 L084G 1.01 85 D085R 1.03 85 D085A 1.09 85 D085H 1.24 85 D085E 1.25 85 D085C 1.50 85 D085G 1.60 85 D085F 1.98 86 L086C 2.44 86 L086A 3.32 87 V087P 1.64 87 V087C 2.22 87 V087L 4.30 88 I088M 1.09 88 I088P 3.51 89 I089L 1.22 89 I089A 1.83 89 I089P 1.91 90 M090C 1.09 90 M090E 1.15 90 M090A 1.41 90 M090D 2.88 91 L091I 1.05 91 L091C 1.27 91 L091A 1.45 91 L091D 1.47 92 G092C 2.05 93 T093A 1.05 96 T096F 1.24 96 T096G 1.28 96 T096L 1.93 96 T096M 2.53 96 T096C 3.76 96 T096A 4.20 98 A098Y 1.15 98 A098P 1.26 98 A098N 1.40 98 A098C 1.42 98 A098L 1.47 98 A098D 2.19 100 F100C 1.28 100 F100T 1.42 100 F100N 1.45 100 F100A 2.02 100 F100M 2.19 101 R101L 1.12 102 R102Q 1.19 102 R102Y 1.29 102 R102L 1.64 102 R102A 1.79 104 P104V 1.02 104 P104H 1.03 104 P104N 1.44 104 P104C 1.83 104 P104E 1.97 104 P104I 2.05 104 P104M 2.24 105 L105Q 1.04 105 L105H 1.23 105 L105R 1.25 105 L105G 1.40 105 L105W 1.71 105 L105F 1.73 105 L105C 1.92 106 D106S 1.02 106 D106W 1.07 106 D106E 1.09 106 D106C 1.10 106 D106A 1.13 106 D106H 1.18 106 D106K 1.24 106 D106T 1.38 106 D106F 1.45 106 D106G 1.45 106 D106V 1.68 107 I107L 1.04 107 I107S 1.33 107 I107C 1.41 107 I107T 1.53 108 A108S 1.00 108 A108G 1.13 108 A108L 2.56 108 A108K 2.97 110 G110A 1.01 110 G110D 1.40 110 G110C 1.43 110 G110E 1.76 110 G110F 2.29 111 M111C 1.01 111 M111A 1.02 111 M111I 1.03 111 M111Y 1.06 111 M111W 1.23 111 M111N 1.31 112 S112L 1.00 112 S112E 1.16 113 V113M 1.06 113 V113Q 1.11 113 V113R 1.11 113 V113P 1.14 113 V113N 1.22 113 V113A 1.31 114 L114T 1.05 114 L114A 1.07 114 L114G 1.14 114 L114C 1.14 114 L114I 1.17 114 L114M 1.28 115 V115C 1.08 115 V115S 1.14 115 V115Q 1.15 115 V115A 1.19 115 V115T 1.28 115 V115L 1.30 115 V115M 1.32 115 V115R 1.63 115 V115F 1.69 115 V115G 1.76 115 V115Y 2.07 115 V115D 2.21 115 V115P 2.21 115 V115W 2.48 116 T116N 1.05 116 T116C 1.05 116 T116H 1.08 116 T116M 1.39 117 Q117F 1.02 117 Q117R 1.05 117 Q117T 1.10 117 Q117H 1.12 117 Q117Y 1.13 117 Q117P 1.13 117 Q117E 1.21 117 Q117A 1.73 117 Q117M 1.89 118 V118L 1.05 118 V118C 1.14 118 V118Y 1.34 118 V118Q 1.50 119 L119A 1.02 120 T120V 1.07 120 T120S 1.07 120 T120K 1.09 120 T120M 1.22 120 T120L 1.26 120 T120N 1.42 120 T120E 1.53 120 T120I 1.56 120 T120Y 1.61 121 S121E 1.04 121 S121N 1.06 121 S121Q 1.09 121 S121T 1.26 121 S121L 1.49 121 S121A 1.55 121 S121V 1.59 121 S121C 1.64 122 A122L 1.02 123 G123K 1.12 123 G123A 1.19 123 G123Y 1.24 123 G123M 1.38 123 G123L 1.38 123 G123W 1.39 125 V125G 1.09 126 G126M 1.17 126 G126D 1.22 127 T127A 1.10 128 T128M 1.06 128 T128H 1.08 128 T128V 1.15 128 T128P 1.16 128 T128W 1.23 128 T128S 1.27 128 T128A 1.31 128 T128Q 1.34 128 T128N 1.36 128 T128K 1.57 128 T128R 1.70 128 T128F 1.71 128 T128L 1.72 128 T128Y 1.81 131 A131R 1.04 132 P132N 1.05 132 P132L 2.24 132 P132E 3.02 132 P132Y 4.78 132 P132G 4.98 132 P132S 5.05 132 P132C 5.68 132 P132A 6.08 132 P132Q 6.15 133 K133Y 1.44 133 K133L 1.92 134 V134C 1.37 134 V134G 1.42 134 V134S 1.44 134 V134L 1.45 134 V134A 1.64 134 V134P 1.71 134 V134M 1.89 134 V134N 2.80 135 L135D 2.90 136 V136T 1.13 136 V136L 1.13 136 V136C 1.23 136 V136A 1.60 137 V137M 1.13 137 V137L 1.27 137 V137C 1.42 137 V137A 1.46 138 S138G 1.11 138 S138C 1.18 138 S138A 1.28 138 S138N 1.31 138 S138P 1.39 140 P140C 1.07 140 P140A 1.83 140 P140H 2.25 140 P140F 2.89 140 P140G 3.11 141 P141A 1.08 143 A143C 1.07 143 A143E 1.13 143 A143D 1.22 143 A143L 1.28 143 A143H 1.36 143 A143K 1.37 144 P144M 1.01 144 P144F 1.08 144 P144Q 1.08 144 P144K 1.09 144 P144R 1.14 144 P144L 1.15 144 P144D 1.38 144 P144N 1.49 144 P144H 1.60 144 P144Y 1.65 146 P146N 1.00 146 P146G 1.04 146 P146R 1.06 146 P146M 1.23 146 P146A 1.36 146 P146Y 1.44 146 P146F 1.53 146 P146H 1.57 146 P146C 1.69 146 P146L 2.00 147 H147Q 1.03 147 H147W 1.05 147 H147K 1.06 147 H147E 1.10 147 H147Y 1.12 147 H147C 1.17 147 H147D 1.18 147 H147P 1.21 147 H147N 1.25 147 H147L 1.29 147 H147M 1.44 148 P148V 1.04 148 P148A 1.06 148 P148T 1.09 148 P148E 1.19 148 P148G 1.20 148 P148S 1.21 148 P148R 1.25 148 P148K 1.30 148 P148D 1.34 148 P148Y 1.37 148 P148L 1.39 148 P148F 1.50 149 W149H 1.01 150 F150Y 1.07 150 F150H 1.18 150 F150L 1.30 151 Q151P 1.91 151 Q151E 2.07 151 Q151K 2.19 151 Q151H 2.19 151 Q151S 2.25 151 Q151R 2.32 151 Q151T 2.37 151 Q151C 2.55 151 Q151Y 2.75 151 Q151D 2.81 151 Q151A 2.93 151 Q151M 6.36 152 L152M 1.10 152 L152C 1.14 152 L152E 1.23 152 L152A 1.29 152 L152Y 1.37 152 L152W 1.55 153 I153V 1.15 153 I153A 1.49 153 I153L 1.50 153 I153T 1.62 153 I153S 1.66 153 I153F 1.75 153 I153P 1.87 153 I153H 2.00 153 I153K 2.44 154 F154Y 4.96 155 E155S 1.12 155 E155G 1.12 155 E155T 1.19 155 E155D 1.24 155 E155K 1.33 155 E155N 1.79 155 E155L 2.07 155 E155A 2.59 155 E155P 2.60 155 E155Y 2.65 155 E155M 2.91 156 G156S 1.04 156 G156K 1.11 156 G156E 1.14 156 G156R 1.21 156 G156A 1.21 156 G156P 1.29 156 G156C 1.37 156 G156N 1.38 156 G156H 1.40 156 G156Y 1.40 156 G156T 1.53 156 G156M 1.62 156 G156D 1.62 157 G157I 1.33 157 G157F 1.42 157 G157K 1.47 157 G157H 1.57 158 E158H 1.01 158 E158P 1.19 158 E158Q 1.24 158 E158S 1.27 158 E158A 1.28 158 E158R 1.29 158 E158W 1.31 158 E158C 1.37 158 E158N 1.58 158 E158M 1.73 158 E158F 1.77 158 E158K 1.88 158 E158L 1.96 158 E158Y 2.48 159 Q159H 1.48 160 K160N 1.12 160 K160A 1.14 160 K160R 1.15 160 K160D 1.19 160 K160C 1.29 160 K160Q 1.41 160 K160M 1.47 160 K160P 1.66 161 T161L 1.16 161 T161V 1.24 161 T161Q 1.50 161 T161M 1.72 161 T161Y 2.62 162 T162R 1.23 162 T162G 1.82 162 T162S 2.01 162 T162W 2.04 162 T162I 2.21 162 T162Q 2.45 162 T162Y 2.89 162 T162K 3.13 162 T162F 3.23 162 T162M 3.49 162 T162C 3.57 162 T162L 3.59 162 T162N 3.84 162 T162H 3.91 162 T162P 4.37 163 E163N 1.00 163 E163C 1.08 163 E163D 1.08 163 E163A 1.79 163 E163Y 1.89 163 E163L 1.94 164 L164Q 1.01 164 L164V 1.02 164 L164S 1.11 164 L164M 1.26 164 L164N 1.31 164 L164R 1.61 164 L164P 2.41 165 A165G 1.07 165 A165V 1.13 165 A165N 1.20 165 A165R 1.29 165 A165Q 1.32 165 A165T 1.32 165 A165P 1.34 165 A165C 1.42 165 A165L 1.55 165 A165M 1.56 165 A165D 1.69 166 R166W 1.08 166 R166F 1.10 166 R166K 1.20 166 R166N 1.21 166 R166Y 1.22 166 R166M 1.29 166 R166I 1.39 166 R166P 1.50 166 R166L 1.50 166 R166A 1.51 166 R166D 1.55 166 R166H 1.56 167 V167I 1.00 167 V167S 1.86 167 V167H 2.11 167 V167Y 2.15 167 V167R 2.25 167 V167Q 2.41 167 V167T 2.47 167 V167L 2.56 167 V167G 2.83 167 V167M 3.84 167 V167A 4.99 167 V167C 5.37 167 V167D 5.54 167 V167P 6.08 168 Y168F 5.17 168 Y168L 5.39 169 S169Y 1.10 169 S169A 1.13 169 S169R 1.19 169 S169K 1.27 169 S169Q 1.37 169 S169C 1.38 169 S169M 1.40 169 S169L 1.47 169 S169I 1.53 170 A170C 1.06 170 A170E 1.17 170 A170F 1.17 170 A170N 1.17 170 A170M 1.28 170 A170D 1.32 170 A170P 1.33 171 L171H 1.07 171 L171G 1.33 171 L171Y 1.35 171 L171T 1.36 171 L171V 1.39 171 L171I 1.42 171 L171K 1.53 171 L171A 1.66 171 L171C 1.73 171 L171S 1.76 171 L171Q 1.93 171 L171F 1.97 171 L171M 2.22 171 L171N 2.79 172 A172M 1.06 172 A172L 1.22 172 A172D 1.42 172 A172Y 1.76 173 S173T 1.29 173 S173H 1.49 173 S173I 2.22 173 S173F 2.30 173 S173R 2.47 173 S173V 2.54 173 S173E 2.65 173 S173P 2.66 173 S173A 2.72 173 S173M 3.01 173 S173K 3.01 173 S173C 3.07 173 S173Y 3.54 173 S173W 3.67 173 S173L 3.86 174 F174H 1.05 174 F174K 1.17 174 F174P 1.46 174 F174Y 1.66 174 F174L 1.83 174 F174A 2.09 174 F174M 2.20 175 M175N 1.02 175 M175E 1.43 176 K176C 1.01 176 K176R 1.03 176 K176E 1.08 176 K176W 1.16 176 K176D 1.18 176 K176A 1.19 176 K176F 1.28 176 K176V 1.33 176 K176M 1.33 178 P178K 1.70 178 P178T 2.28 178 P178V 2.70 178 P178G 2.95 178 P178S 3.06 178 P178Q 3.64 178 P178M 3.87 178 P178E 4.15 178 P178A 4.39 178 P178D 6.44 178 P178Y 6.91 178 P178L 7.15 179 F179G 1.16 179 F179V 1.17 179 F179Y 1.47 179 F179E 1.80 179 F179L 1.89 180 F180W 1.81 180 F180C 1.94 180 F180I 2.11 180 F180L 2.13 180 F180A 2.70 180 F180Y 2.99 180 F180N 3.05 180 F180V 3.24 180 F180M 4.36 181 D181A 1.23 183 G183P 1.02 183 G183R 1.09 183 G183Y 1.45 183 G183L 1.50 183 G183C 1.99 184 S184Y 1.09 184 S184Q 1.16 184 S184I 1.21 184 S184V 1.25 184 S184F 1.27 184 S184K 1.61 184 S184A 1.69 184 S184M 1.77 184 S184E 1.86 184 S184N 1.93 184 S184L 2.00 184 S184D 2.24 184 S184C 2.39 185 V185F 1.20 185 V185Q 1.41 185 V185M 1.46 186 I186L 1.14 186 I186M 1.38 186 I186A 1.79 186 I186D 4.29 187 S187K 1.16 187 S187D 1.40 187 S187G 1.46 187 S187L 1.46 187 S187H 1.51 187 S187I 1.58 187 S187N 1.59 187 S187C 1.67 187 S187A 1.72 187 S187M 1.87 188 T188N 1.69 188 T188E 1.97 189 D189A 1.18 189 D189T 1.21 189 D189I 1.27 189 D189L 1.30 190 G190C 1.17 190 G190Y 1.39 190 G190P 1.86 190 G190D 2.02 190 G190H 2.92 190 G190A 3.42 190 G190M 5.54 191 V191T 1.03 191 V191R 1.91 191 V191K 2.17 191 V191F 2.75 191 V191C 2.81 191 V191Y 4.34 191 V191L 4.69 191 V191A 5.06 191 V191E 5.46 191 V191Q 5.83 191 V191D 6.03 191 V191M 7.34 193 G193S 1.60 193 G193E 3.15 193 G193Q 4.29 193 G193V 5.21 195 H195P 1.16 195 H195M 1.28 195 H195K 1.33 195 H195Y 1.49 195 H195E 1.70 195 H195D 1.93 196 F196I 1.12 196 F196L 1.17 196 F196C 1.18 197 T197H 1.24 197 T197A 1.42 197 T197M 2.38 198 E198T 1.16 198 E198S 1.18 198 E198F 1.21 198 E198V 1.44 198 E198Q 1.46 198 E198A 1.46 198 E198I 1.48 198 E198L 1.54 198 E198N 1.67 198 E198P 1.72 198 E198Y 1.77 198 E198W 1.78 198 E198C 1.83 198 E198M 1.86 198 E198R 1.88 199 A199F 1.15 199 A199H 1.15 199 A199R 1.17 199 A199T 1.22 199 A199E 1.31 199 A199D 1.33 199 A199V 1.45 199 A199K 1.53 199 A199Y 1.59 199 A199L 1.65 199 A199C 2.45 201 N201D 1.64 202 R202M 1.76 202 R202G 1.82 202 R202S 1.84 202 R202C 1.93 202 R202A 1.97 202 R202I 1.99 202 R202E 2.05 202 R202L 2.05 202 R202T 2.06 202 R202H 2.09 202 R202F 2.16 202 R202W 2.52 203 D203Q 1.03 203 D203S 1.13 203 D203I 1.19 203 D203N 1.28 203 D203G 1.33 203 D203F 1.34 203 D203H 1.54 203 D203P 1.71 203 D203R 1.77 203 D203A 1.96 203 D203L 2.08 203 D203C 2.09

The following Table provides variants that exhibited peracid degradation that was less than wild-type.

TABLE 10-7 Variants with Peracid Degradation Results Less than Wild-Type Pos WT/Pos./Var. PAD PI 1 M001V 0.94 2 A002Y 0.46 2 A002N 0.59 2 A002V 0.60 2 A002I 0.61 2 A002T 0.61 2 A002S 0.66 2 A002G 0.84 2 A002F 0.93 3 K003V 0.84 4 R004L 0.01 4 R004V 0.08 4 R004I 0.15 4 R004W 0.48 4 R004G 0.79 4 R004S 0.91 4 R004E 0.97 4 R004Y 0.98 4 R004H 0.99 4 R004Q 0.99 4 R004T 1.00 5 I005G 0.01 5 I005N 0.01 5 I005P 0.01 5 I005R 0.01 5 I005F 0.15 5 I005S 0.37 5 I005H 0.63 5 I005T 0.72 5 I005V 0.92 6 L006S 0.01 6 L006K 0.01 6 L006G 0.01 6 L006H 0.01 6 L006R 0.01 6 L006W 0.01 6 L006E 0.01 6 L006Q 0.01 6 L006V 0.35 6 L006T 0.35 6 L006I 0.82 7 C007S 0.01 7 C007R 0.01 7 C007Y 0.54 7 C007M 0.68 7 C007G 0.69 8 F008S 0.01 8 F008R 0.46 8 F008H 0.64 8 F008G 0.65 8 F008T 0.77 8 F008K 0.83 8 F008P 0.83 8 F008V 0.85 8 F008Y 0.90 8 F008N 0.96 9 G009H 0.01 9 G009T 0.01 10 D010W 0.01 10 D010K 0.01 10 D010Y 0.01 10 D010T 0.01 10 D010I 0.01 10 D010V 0.01 10 D010S 0.01 10 D010G 0.01 10 D010R 0.01 10 D010A 0.01 10 D010M 0.01 10 D010N 0.01 10 D010P 0.01 10 D010E 0.15 11 S011T 0.01 11 S011V 0.01 11 S011D 0.01 11 S011E 0.01 11 S011F 0.01 11 S011G 0.01 11 S011L 0.01 11 S011Q 0.01 11 S011R 0.01 11 S011H 0.33 11 S011K 0.40 11 S011A 0.53 11 S011I 0.56 12 L012V 0.01 12 L012S 0.01 12 L012G 0.01 12 L012R 0.01 12 L012D 0.01 12 L012P 0.01 12 L012W 0.02 12 L012T 0.06 12 L012A 0.07 12 L012K 0.13 12 L012H 0.16 12 L012F 0.17 12 L012Q 0.22 12 L012C 0.22 12 L012N 0.66 13 T013Q 0.51 13 T013V 0.63 13 T013S 0.68 13 T013G 0.77 14 W014I 0.01 14 W014S 0.01 14 W014G 0.01 14 W014K 0.01 14 W014V 0.01 14 W014L 0.01 14 W014T 0.01 14 W014R 0.01 14 W014N 0.01 14 W014P 0.01 14 W014E 0.15 14 W014F 0.22 14 W014A 0.27 14 W014Y 0.66 15 G015C 0.01 15 G015N 0.01 15 G015D 0.01 15 G015E 0.01 15 G015P 0.01 15 G015A 0.61 15 G015S 0.63 16 W016S 0.01 16 W016G 0.01 16 W016H 0.01 16 W016T 0.01 16 W016R 0.01 16 W016N 0.01 16 W016P 0.15 16 W016Q 0.31 16 W016M 0.37 16 W016A 0.55 16 W016D 0.57 16 W016E 0.65 16 W016V 0.88 17 V017A 0.68 17 V017E 0.75 17 V017G 0.84 17 V017K 0.84 17 V017F 0.85 17 V017T 0.86 17 V017Y 0.88 17 V017R 0.94 17 V017P 0.96 17 V017I 0.99 17 V017L 1.00 18 P018S 0.07 19 V019P 0.01 19 V019M 0.12 19 V019R 0.34 19 V019Q 0.40 19 V019A 0.55 19 V019G 0.56 19 V019S 0.57 19 V019E 0.62 19 V019Y 0.70 19 V019D 0.79 19 V019L 0.91 19 V019K 0.97 20 E020L 0.73 20 E020G 0.78 21 D021P 0.86 22 G022K 0.01 22 G022W 0.23 22 G022R 0.56 22 G022V 0.85 22 G022S 0.98 23 A023R 0.28 23 A023S 0.34 23 A023G 0.35 23 A023F 0.44 23 A023V 0.60 23 A023Q 0.73 23 A023P 0.73 23 A023W 0.80 23 A023M 0.95 23 A023Y 0.96 24 P024S 0.61 24 P024Q 0.65 24 P024T 0.66 24 P024A 0.68 24 P024G 0.76 24 P024I 0.85 24 P024R 0.91 24 P024H 0.97 25 T025P 0.01 25 T025H 0.01 25 T025L 0.01 25 T025R 0.01 25 T025M 0.01 25 T025E 0.01 25 T025D 0.01 25 T025K 0.13 25 T025W 0.14 25 T025I 0.35 25 T025G 0.43 25 T025C 0.51 25 T025V 0.51 25 T025S 0.58 25 T025A 0.86 26 E026S 0.28 26 E026T 0.40 26 E026W 0.47 26 E026N 0.48 26 E026R 0.81 26 E026G 0.87 26 E026C 0.94 26 E026V 0.97 26 E026P 0.99 27 R027W 0.01 27 R027T 0.01 27 R027P 0.48 27 R027C 0.58 27 R027S 0.69 27 R027G 0.84 27 R027E 0.93 27 R027V 0.94 28 F028G 0.01 28 F028P 0.39 28 F028V 0.53 28 F028S 0.70 29 A029V 0.44 29 A029T 0.47 29 A029S 0.55 29 A029Y 0.59 29 A029P 0.62 29 A029R 0.73 29 A029W 0.74 29 A029M 0.77 29 A029G 0.80 29 A029E 0.84 29 A029D 1.00 30 P030M 0.79 30 P030Q 0.91 30 P030A 0.92 31 D031E 0.88 32 V032P 0.01 32 V032R 0.72 33 R033V 0.94 34 W034R 0.01 34 W034E 0.01 34 W034Q 0.04 34 W034S 0.08 34 W034T 0.15 34 W034V 0.73 34 W034G 0.88 34 W034I 0.94 35 T035Q 0.01 35 T035N 0.01 35 T035R 0.01 35 T035V 0.34 36 G036S 0.26 36 G036T 0.33 36 G036V 0.38 36 G036M 0.54 36 G036N 0.56 36 G036W 0.68 36 G036Q 0.71 36 G036R 0.90 37 V037T 0.81 37 V037H 0.96 37 V037W 0.98 38 L038K 0.01 38 L038G 0.01 38 L038E 0.01 38 L038P 0.01 38 L038Q 0.01 38 L038R 0.01 38 L038D 0.12 38 L038S 0.29 38 L038A 0.63 38 L038C 0.72 39 A039S 0.01 39 A039G 0.30 39 A039N 0.43 39 A039R 0.64 39 A039I 0.71 39 A039P 0.74 39 A039T 0.79 39 A039M 0.81 39 A039E 0.83 39 A039C 0.92 39 A039K 0.96 39 A039L 0.97 39 A039V 0.98 40 Q040P 0.01 41 Q041V 0.01 41 Q041S 0.22 41 Q041P 0.66 41 Q041Y 0.70 41 Q041W 0.88 42 L042W 0.01 42 L042H 0.01 42 L042T 0.01 42 L042Q 0.28 42 L042S 0.45 42 L042R 0.64 42 L042I 0.66 42 L042V 0.73 42 L042M 0.74 42 L042G 0.76 43 G043S 0.23 43 G043P 0.31 43 G043V 0.33 43 G043Q 0.48 43 G043R 0.59 43 G043C 0.73 43 G043I 0.77 43 G043K 0.86 43 G043M 0.88 43 G043Y 0.94 43 G043H 0.96 44 A044S 0.01 44 A044Y 0.01 44 A044T 0.01 44 A044R 0.01 44 A044E 0.03 44 A044V 0.50 44 A044F 0.80 44 A044W 0.85 44 A044M 0.98 44 A044L 0.99 45 D045S 0.38 45 D045T 0.44 45 D045R 0.49 45 D045V 0.50 45 D045P 0.53 45 D045Q 0.57 45 D045W 0.58 45 D045H 0.78 45 D045L 0.78 45 D045M 0.78 45 D045G 0.84 45 D045A 0.84 45 D045C 0.84 45 D045K 0.87 46 F046T 0.43 46 F046W 0.63 46 F046S 0.66 46 F046V 0.79 46 F046I 0.88 46 F046G 0.94 47 E047P 0.36 47 E047R 0.62 47 E047N 0.63 47 E047S 0.63 47 E047M 0.70 47 E047A 0.76 47 E047F 0.76 47 E047C 0.77 47 E047T 0.84 47 E047D 0.98 47 E047H 0.99 48 V048R 0.01 48 V048S 0.42 48 V048G 0.87 48 V048N 0.98 48 V048E 0.99 49 I049P 0.16 49 I049R 0.29 49 I049W 0.68 49 I049H 0.74 49 I049S 0.79 49 I049E 0.88 49 I049V 0.97 50 E050R 0.01 50 E050W 0.14 50 E050V 0.43 50 E050I 0.58 50 E050S 0.65 50 E050Q 0.91 50 E050L 0.97 51 E051R 0.01 51 E051I 0.04 51 E051W 0.17 51 E051V 0.37 51 E051Q 0.76 51 E051L 0.93 52 G052H 0.01 52 G052S 0.01 52 G052V 0.01 52 G052T 0.01 52 G052M 0.01 52 G052F 0.01 52 G052I 0.07 52 G052P 0.24 52 G052L 0.24 52 G052Q 0.28 52 G052R 0.35 52 G052E 0.55 52 G052A 0.79 53 L053R 0.01 53 L053W 0.01 53 L053P 0.01 53 L053D 0.01 53 L053E 0.19 53 L053K 0.24 53 L053S 0.26 53 L053G 0.33 53 L053V 0.65 53 L053I 0.66 53 L053Q 0.72 53 L053T 0.84 54 S054F 0.01 54 S054W 0.01 54 S054H 0.01 54 S054K 0.08 54 S054I 0.12 54 S054Y 0.12 54 S054G 0.17 54 S054L 0.26 54 S054V 0.29 54 S054E 0.30 54 S054T 0.33 54 S054R 0.35 54 S054M 0.48 54 S054Q 0.53 54 S054D 0.65 54 S054C 0.88 55 A055V 0.01 55 A055I 0.01 55 A055P 0.01 55 A055W 0.01 55 A055Y 0.18 55 A055R 0.25 55 A055T 0.42 55 A055G 0.73 55 A055L 0.87 55 A055S 0.87 55 A055H 0.92 56 R056C 0.01 56 R056G 0.01 56 R056T 0.01 56 R056E 0.01 56 R056Q 0.01 56 R056S 0.12 56 R056L 0.24 56 R056N 0.27 56 R056A 0.69 57 T057R 0.01 57 T057P 0.01 57 T057N 0.25 57 T057C 0.40 57 T057Y 0.55 57 T057H 0.61 57 T057A 0.65 57 T057L 0.76 57 T057V 0.87 57 T057I 0.87 58 T058M 0.03 58 T058A 0.36 58 T058V 0.96 58 T058S 0.96 59 N059R 0.01 59 N059M 0.01 59 N059P 0.01 60 I060P 0.32 60 I060D 0.66 60 I060C 0.67 60 I060M 0.68 60 I060A 0.79 60 I060R 0.81 60 I060L 0.91 60 I060E 0.92 60 I060K 0.96 60 I060S 1.00 61 D061F 0.70 61 D061A 0.71 61 D061C 0.85 61 D061Y 0.95 61 D061V 0.97 61 D061N 1.00 62 D062T 0.01 62 D062I 0.01 62 D062V 0.01 62 D062H 0.01 62 D062W 0.01 62 D062S 0.01 62 D062L 0.01 62 D062G 0.01 62 D062R 0.01 62 D062M 0.01 62 D062P 0.01 62 D062Q 0.01 62 D062A 0.11 62 D062C 0.49 62 D062E 0.60 63 P063A 0.60 63 P063R 0.80 63 P063S 0.90 63 P063M 0.91 63 P063F 0.93 63 P063Y 0.95 64 T064R 0.11 64 T064D 0.64 64 T064W 0.69 64 T064Q 0.87 64 T064C 0.88 64 T064P 0.94 64 T064H 0.96 64 T064N 0.98 64 T064S 0.99 65 D065V 0.20 65 D065R 0.22 65 D065H 0.40 65 D065Y 0.42 65 D065P 0.42 65 D065S 0.47 65 D065W 0.50 65 D065T 0.50 65 D065G 0.52 65 D065I 0.62 65 D065A 0.72 66 P066N 0.38 66 P066Q 0.42 66 P066G 0.44 66 P066R 0.51 66 P066C 0.52 66 P066A 0.56 66 P066F 0.67 66 P066Y 0.70 66 P066D 0.72 66 P066I 0.84 66 P066V 0.89 66 P066H 0.95 66 P066L 0.99 67 R067F 0.01 67 R067W 0.02 67 R067P 0.04 67 R067E 0.11 67 R067V 0.12 67 R067Q 0.13 67 R067L 0.16 67 R067A 0.22 67 R067T 0.32 67 R067N 0.33 67 R067G 0.41 67 R067K 0.99 68 L068G 0.01 68 L068A 0.01 68 L068M 0.03 68 L068C 0.06 68 L068S 0.07 68 L068N 0.10 68 L068E 0.13 68 L068H 0.22 68 L068Q 0.25 68 L068F 0.25 68 L068T 0.32 68 L068P 0.35 68 L068D 0.44 68 L068Y 0.45 68 L068R 0.47 68 L068V 0.51 68 L068W 0.56 68 L068I 0.73 69 N069Y 0.17 69 N069W 0.55 69 N069P 0.59 69 N069R 0.83 69 N069G 0.98 70 G070M 0.01 70 G070T 0.01 70 G070P 0.01 70 G070V 0.01 70 G070C 0.01 70 G070R 0.01 70 G070Y 0.01 70 G070K 0.01 70 G070N 0.01 70 G070Q 0.01 70 G070F 0.01 70 G070I 0.27 70 G070E 0.33 70 G070S 0.64 71 A071P 0.01 71 A071N 0.61 71 A071D 0.65 71 A071G 0.68 71 A071S 0.69 71 A071R 0.77 71 A071H 0.78 71 A071I 0.79 71 A071T 0.79 71 A071E 0.81 71 A071L 0.84 71 A071F 0.99 71 A071C 0.99 72 S072Y 0.07 72 S072W 0.34 72 S072P 0.56 72 S072Q 0.66 72 S072L 0.70 72 S072R 0.74 72 S072D 0.80 72 S072V 0.83 72 S072E 0.93 72 S072T 0.97 73 Y073P 0.01 73 Y073R 0.26 73 Y073L 0.50 73 Y073G 0.51 73 Y073H 0.52 73 Y073I 0.64 73 Y073S 0.68 73 Y073V 0.74 73 Y073N 0.76 73 Y073D 0.80 73 Y073Q 0.87 73 Y073K 0.94 74 L074S 0.01 74 L074G 0.57 74 L074V 0.61 74 L074I 0.64 74 L074W 0.67 74 L074Y 0.86 75 P075M 0.30 75 P075R 0.46 75 P075Q 0.61 75 P075S 0.63 75 P075T 0.69 75 P075I 0.74 75 P075H 0.86 75 P075K 0.88 75 P075G 0.93 76 S076W 0.01 76 S076Y 0.18 76 S076F 0.46 76 S076Q 0.90 77 C077Y 0.01 77 C077R 0.01 77 C077W 0.01 77 C077F 0.01 77 C077G 0.18 77 C077L 0.73 77 C077S 0.76 77 C077V 0.80 77 C077A 0.91 78 L078E 0.01 78 L078N 0.01 78 L078M 0.48 78 L078Q 0.52 78 L078C 0.78 78 L078Y 0.81 78 L078V 0.83 79 A079H 0.01 79 A079F 0.01 79 A079C 0.03 79 A079Q 0.27 79 A079E 0.27 79 A079N 0.28 79 A079M 0.28 79 A079R 0.32 79 A079W 0.53 79 A079T 0.60 79 A079I 0.67 79 A079S 0.78 79 A079G 0.92 79 A079P 0.94 79 A079L 0.96 80 T080W 0.01 80 T080L 0.01 80 T080K 0.01 80 T080R 0.01 80 T080E 0.01 80 T080P 0.01 80 T080H 0.05 80 T080Y 0.11 80 T080I 0.15 80 T080N 0.53 81 H081R 0.01 81 H081Y 0.14 81 H081K 0.56 81 H081S 0.69 81 H081V 0.71 81 H081P 0.72 81 H081Q 0.75 81 H081G 0.80 81 H081F 0.90 82 L082R 0.01 82 L082S 0.01 82 L082W 0.01 82 L082V 0.19 82 L082G 0.31 82 L082T 0.38 82 L082H 0.47 82 L082I 0.51 82 L082K 0.51 82 L082P 0.52 82 L082A 0.98 83 P083T 0.01 83 P083V 0.19 83 P083L 0.21 83 P083H 0.61 83 P083W 0.62 83 P083G 0.68 83 P083S 0.79 83 P083Q 0.82 83 P083D 0.83 83 P083F 0.99 84 L084W 0.01 84 L084V 0.42 84 L084P 0.43 84 L084T 0.44 84 L084A 0.45 84 L084Q 0.52 84 L084S 0.55 84 L084R 0.57 84 L084N 0.67 84 L084K 0.79 84 L084D 0.85 84 L084I 0.87 84 L084H 0.99 85 D085I 0.10 85 D085L 0.24 85 D085V 0.25 85 D085W 0.34 85 D085P 0.54 85 D085Y 0.55 85 D085S 0.68 85 D085T 0.71 85 D085N 0.78 85 D085Q 0.99 86 L086H 0.01 86 L086S 0.01 86 L086R 0.01 86 L086E 0.01 86 L086Q 0.01 86 L086W 0.08 86 L086V 0.12 86 L086T 0.28 86 L086G 0.70 86 L086Y 0.82 86 L086P 0.99 87 V087S 0.01 87 V087G 0.01 87 V087Y 0.01 87 V087R 0.01 87 V087K 0.01 87 V087D 0.01 87 V087F 0.10 87 V087T 0.15 87 V087A 0.17 87 V087M 0.75 88 I088H 0.01 88 I088T 0.01 88 I088G 0.01 88 I088N 0.01 88 I088Q 0.01 89 I089H 0.01 89 I089S 0.01 89 I089G 0.01 89 I089W 0.01 89 I089Q 0.01 89 I089E 0.01 89 I089F 0.75 89 I089V 0.82 89 I089T 0.90 90 M090S 0.01 90 M090W 0.01 90 M090G 0.01 90 M090P 0.01 90 M090V 0.08 90 M090T 0.15 90 M090R 0.36 90 M090I 0.66 90 M090Q 0.77 90 M090L 0.98 91 L091G 0.01 91 L091T 0.01 91 L091Q 0.01 91 L091E 0.01 91 L091S 0.43 91 L091V 0.79 91 L091M 0.88 92 G092V 0.01 92 G092S 0.01 92 G092E 0.01 92 G092F 0.01 93 T093Q 0.01 93 T093Y 0.03 93 T093D 0.23 93 T093S 0.49 93 T093F 0.54 93 T093C 0.95 94 N094L 0.01 94 N094T 0.01 94 N094V 0.01 94 N094H 0.01 94 N094R 0.01 94 N094W 0.01 94 N094M 0.03 94 N094C 0.07 94 N094Y 0.12 94 N094G 0.53 94 N094A 0.74 94 N094P 0.79 94 N094S 0.88 95 D095E 0.75 96 T096I 0.01 96 T096W 0.01 96 T096Y 0.01 96 T096R 0.14 96 T096V 0.59 96 T096S 0.79 96 T096P 0.89 97 K097Q 0.01 97 K097G 0.01 97 K097I 0.01 97 K097W 0.01 97 K097L 0.01 97 K097V 0.01 97 K097Y 0.01 97 K097S 0.01 97 K097T 0.01 97 K097M 0.22 97 K097A 0.23 97 K097P 0.27 97 K097R 0.59 98 A098T 0.27 98 A098G 0.56 98 A098S 0.65 98 A098I 0.65 98 A098H 0.92 99 Y099R 0.29 99 Y099V 0.31 99 Y099S 0.37 99 Y099W 0.57 99 Y099H 0.59 99 Y099I 0.61 99 Y099G 0.70 99 Y099P 0.81 99 Y099A 0.82 99 Y099L 0.86 100 F100W 0.01 100 F100K 0.01 100 F100D 0.01 100 F100E 0.15 100 F100S 0.85 101 R101W 0.01 101 R101K 0.07 101 R101Q 0.11 101 R101V 0.44 101 R101D 0.80 101 R101Y 0.80 101 R101P 0.86 101 R101N 0.92 101 R101C 0.95 101 R101I 0.96 101 R101F 0.97 102 R102W 0.01 102 R102F 0.23 102 R102G 0.27 102 R102C 0.36 102 R102V 0.61 102 R102D 0.68 102 R102P 0.89 102 R102S 0.96 103 T103W 0.01 103 T103Y 0.01 103 T103G 0.01 103 T103K 0.01 103 T103I 0.01 103 T103L 0.01 103 T103H 0.01 103 T103A 0.01 103 T103V 0.01 103 T103S 0.01 103 T103C 0.01 103 T103R 0.01 103 T103N 0.01 103 T103F 0.01 103 T103P 0.01 104 P104R 0.01 104 P104W 0.23 104 P104T 0.33 104 P104S 0.53 104 P104Q 0.85 104 P104F 0.86 104 P104G 0.98 105 L105V 0.01 105 L105E 0.53 105 L105S 0.61 105 L105Y 0.62 105 L105T 0.64 105 L105P 0.90 106 D106R 0.56 106 D106Q 0.62 106 D106P 0.63 106 D106N 0.64 106 D106M 0.86 106 D106I 0.92 106 D106L 1.00 107 I107E 0.01 107 I107G 0.01 107 I107F 0.01 107 I107Q 0.01 107 I107R 0.01 107 I107P 0.32 107 I107Y 0.52 107 I107A 0.80 107 I107N 0.93 107 I107V 0.97 108 A108E 0.61 108 A108Q 0.73 108 A108T 0.87 108 A108V 0.95 109 L109W 0.01 109 L109D 0.11 109 L109I 0.14 109 L109E 0.19 109 L109R 0.21 109 L109H 0.22 109 L109Q 0.22 109 L109F 0.32 109 L109A 0.32 109 L109S 0.38 109 L109P 0.43 109 L109G 0.51 109 L109V 0.54 109 L109M 0.63 109 L109N 0.66 109 L109T 0.79 109 L109Y 0.83 110 G110T 0.01 110 G110W 0.01 110 G110Y 0.01 110 G110P 0.22 110 G110I 0.23 110 G110S 0.30 110 G110Q 0.34 110 G110R 0.48 110 G110H 0.73 110 G110N 0.77 110 G110M 0.82 111 M111R 0.01 111 M111S 0.14 111 M111H 0.19 111 M111G 0.32 111 M111P 0.57 111 M111E 0.67 111 M111L 0.67 111 M111K 0.71 111 M111T 0.76 111 M111F 0.78 111 M111D 0.79 111 M111V 0.93 112 S112Y 0.01 112 S112R 0.01 112 S112P 0.01 112 S112H 0.38 112 S112V 0.48 112 S112M 0.56 112 S112W 0.58 112 S112K 0.68 112 S112T 0.72 112 S112N 0.85 112 S112F 0.88 112 S112A 0.94 113 V113S 0.57 113 V113G 0.58 113 V113K 0.72 113 V113H 0.76 113 V113W 0.80 113 V113L 0.85 113 V113T 0.86 113 V113D 0.87 113 V113E 0.94 113 V113C 0.94 113 V113F 0.96 113 V113Y 0.98 114 L114H 0.01 114 L114E 0.01 114 L114Q 0.12 114 L114P 0.28 114 L114S 0.55 114 L114V 0.60 114 L114N 0.77 115 V115I 0.99 116 T116Y 0.47 116 T116V 0.57 116 T116R 0.62 116 T116L 0.68 116 T116W 0.75 116 T116I 0.76 116 T116Q 0.77 116 T116P 0.84 116 T116G 0.90 116 T116E 0.91 116 T116A 0.95 116 T116S 0.96 117 Q117W 0.71 117 Q117V 0.76 117 Q117G 0.79 117 Q117S 0.87 118 V118K 0.01 118 V118W 0.01 118 V118E 0.01 118 V118R 0.07 118 V118P 0.22 118 V118D 0.40 118 V118I 0.55 118 V118G 0.56 118 V118S 0.82 118 V118A 0.85 118 V118T 0.92 118 V118M 0.93 118 V118F 1.00 119 L119G 0.01 119 L119S 0.01 119 L119F 0.01 119 L119R 0.01 119 L119P 0.01 119 L119T 0.10 119 L119N 0.11 119 L119V 0.15 119 L119W 0.20 119 L119C 0.24 119 L119D 0.28 119 L119E 0.32 119 L119I 0.43 119 L119H 0.46 119 L119Y 0.56 120 T120P 0.01 120 T120H 0.50 120 T120R 0.60 120 T120A 0.66 120 T120Q 0.78 120 T120C 0.92 121 S121P 0.38 121 S121R 0.70 121 S121W 0.77 121 S121K 0.78 121 S121G 0.99 122 A122G 0.01 122 A122D 0.06 122 A122F 0.15 122 A122H 0.17 122 A122R 0.40 122 A122S 0.43 122 A122K 0.45 122 A122E 0.47 122 A122T 0.52 122 A122P 0.55 122 A122I 0.65 122 A122N 0.70 122 A122Q 0.74 122 A122W 0.86 122 A122V 0.89 122 A122M 0.94 123 G123C 0.30 123 G123Q 0.31 123 G123T 0.54 123 G123E 0.56 123 G123V 0.59 123 G123R 0.60 123 G123N 0.71 123 G123H 0.74 123 G123F 0.80 123 G123P 0.81 123 G123D 0.84 124 G124I 0.01 124 G124H 0.01 124 G124M 0.01 124 G124W 0.01 124 G124P 0.01 124 G124A 0.03 124 G124Q 0.21 124 G124T 0.32 124 G124V 0.33 124 G124R 0.41 124 G124L 0.54 124 G124S 0.56 124 G124Y 0.56 124 G124N 0.60 124 G124D 0.64 124 G124C 0.67 124 G124F 0.95 125 V125W 0.25 125 V125E 0.39 125 V125R 0.47 125 V125C 0.54 125 V125D 0.54 125 V125P 0.62 125 V125F 0.63 125 V125S 0.79 125 V125Y 0.81 125 V125A 0.93 125 V125I 0.94 126 G126I 0.01 126 G126V 0.18 126 G126Y 0.23 126 G126L 0.54 126 G126A 0.55 126 G126E 0.60 126 G126P 0.67 126 G126T 0.74 126 G126R 0.76 126 G126N 0.85 126 G126S 0.90 126 G126C 0.98 127 T127L 0.01 127 T127E 0.01 127 T127Q 0.15 127 T127I 0.20 127 T127H 0.60 127 T127D 0.62 127 T127M 0.64 127 T127C 0.65 127 T127V 0.68 127 T127G 0.71 127 T127P 0.77 127 T127S 0.83 128 T128D 0.66 129 Y129W 0.01 129 Y129G 0.01 129 Y129K 0.01 129 Y129V 0.01 129 Y129T 0.14 129 Y129A 0.17 129 Y129R 0.18 129 Y129M 0.21 129 Y129D 0.23 129 Y129L 0.27 129 Y129N 0.53 129 Y129P 0.59 129 Y129C 0.61 129 Y129S 0.69 129 Y129F 0.71 130 P130T 0.01 130 P130H 0.01 130 P130G 0.01 130 P130S 0.01 130 P130L 0.09 130 P130E 0.22 130 P130W 0.28 130 P130V 0.37 130 P130I 0.41 130 P130A 0.44 130 P130F 0.48 130 P130R 0.53 130 P130K 0.55 130 P130C 0.64 130 P130M 0.76 131 A131W 0.01 131 A131D 0.40 131 A131Y 0.48 131 A131L 0.59 131 A131S 0.68 131 A131P 0.71 131 A131Q 0.74 131 A131V 0.78 131 A131H 0.82 131 A131G 0.87 131 A131E 0.97 132 P132V 0.01 132 P132T 0.01 132 P132W 0.01 132 P132F 0.01 132 P132I 0.01 132 P132H 0.01 132 P132R 0.01 132 P132D 0.01 133 K133C 0.01 133 K133A 0.10 133 K133V 0.23 133 K133G 0.31 133 K133H 0.31 133 K133M 0.33 133 K133T 0.39 133 K133I 0.45 133 K133Q 0.52 133 K133S 0.58 133 K133F 0.59 133 K133P 0.71 133 K133E 0.76 133 K133R 0.83 133 K133W 0.99 134 V134Q 0.79 134 V134T 0.86 134 V134I 0.89 135 L135T 0.01 135 L135W 0.01 135 L135K 0.01 135 L135S 0.01 135 L135F 0.01 135 L135G 0.01 135 L135R 0.01 135 L135P 0.01 135 L135Q 0.17 135 L135V 0.43 135 L135E 0.63 135 L135M 0.78 136 V136P 0.01 136 V136E 0.20 136 V136N 0.40 137 V137N 0.01 137 V137G 0.26 137 V137S 0.29 137 V137I 0.70 137 V137T 0.93 138 S138I 0.35 138 S138V 0.69 139 P139S 0.01 139 P139G 0.01 139 P139R 0.01 139 P139C 0.01 139 P139D 0.01 139 P139E 0.01 139 P139F 0.01 139 P139H 0.01 139 P139I 0.01 139 P139K 0.01 139 P139N 0.01 139 P139Q 0.01 139 P139T 0.01 139 P139V 0.01 140 P140T 0.01 140 P140S 0.01 140 P140V 0.01 140 P140W 0.01 140 P140I 0.01 140 P140Y 0.01 140 P140Q 0.01 140 P140R 0.01 141 P141R 0.01 141 P141G 0.01 141 P141S 0.02 141 P141T 0.12 141 P141V 0.16 141 P141Q 0.37 141 P141I 0.38 141 P141L 0.65 141 P141H 0.79 141 P141N 0.97 142 L142W 0.01 142 L142I 0.28 142 L142S 0.31 142 L142Q 0.33 142 L142V 0.33 142 L142P 0.44 142 L142F 0.54 142 L142A 0.56 142 L142K 0.66 142 L142C 0.70 143 A143W 0.01 143 A143P 0.39 143 A143G 0.42 143 A143S 0.63 143 A143F 0.68 143 A143Q 0.81 143 A143N 0.82 143 A143T 0.97 143 A143R 0.99 143 A143V 0.99 144 P144G 0.62 144 P144A 0.79 144 P144T 0.81 144 P144S 0.92 145 M145W 0.01 145 M145G 0.26 145 M145E 0.48 145 M145I 0.53 145 M145Q 0.57 145 M145L 0.61 145 M145V 0.63 145 M145R 0.69 145 M145F 0.77 145 M145P 0.78 145 M145S 0.78 145 M145T 0.79 145 M145A 0.79 145 M145Y 0.82 145 M145C 0.93 146 P146W 0.68 146 P146T 0.76 146 P146V 0.77 146 P146S 0.96 147 H147S 0.75 147 H147T 0.84 147 H147I 0.92 147 H147V 0.92 147 H147R 0.94 147 H147A 0.98 148 P148Q 0.98 149 W149R 0.01 149 W149E 0.01 149 W149P 0.01 149 W149C 0.12 149 W149I 0.24 149 W149A 0.31 149 W149S 0.33 149 W149Q 0.40 149 W149T 0.44 149 W149G 0.45 149 W149M 0.49 149 W149F 0.50 149 W149L 0.64 149 W149Y 0.75 150 F150P 0.32 150 F150N 0.36 150 F150G 0.46 150 F150V 0.51 150 F150A 0.54 150 F150T 0.58 150 F150W 0.62 150 F150M 0.63 150 F150E 0.73 150 F150C 0.78 150 F150I 0.78 150 F150K 0.85 151 Q151L 0.01 151 Q151V 0.01 151 Q151F 0.01 151 Q151I 0.01 151 Q151W 0.32 152 L152I 0.61 152 L152P 0.61 152 L152T 0.69 152 L152Q 0.76 152 L152G 0.77 152 L152S 0.84 152 L152D 0.86 152 L152V 0.88 152 L152R 0.91 152 L152K 0.91 152 L152H 0.92 153 I153N 0.89 154 F154T 0.01 154 F154G 0.01 154 F154V 0.01 154 F154S 0.29 154 F154Q 0.97 155 E155R 0.01 155 E155F 0.23 155 E155V 0.47 155 E155I 0.65 155 E155Q 0.69 156 G156I 0.01 156 G156F 0.73 156 G156W 0.90 156 G156L 0.94 156 G156V 0.97 157 G157R 0.01 157 G157P 0.01 157 G157S 0.19 157 G157V 0.40 157 G157C 0.61 157 G157E 0.84 157 G157M 0.85 157 G157A 0.87 157 G157D 0.94 157 G157T 0.99 158 E158V 0.89 158 E158D 0.89 158 E158T 0.91 158 E158I 0.94 159 Q159A 0.28 159 Q159C 0.31 159 Q159P 0.49 159 Q159D 0.63 159 Q159L 0.70 159 Q159G 0.72 159 Q159S 0.73 159 Q159R 0.74 159 Q159M 0.84 159 Q159E 0.97 160 K160W 0.01 160 K160G 0.30 160 K160H 0.57 160 K160S 0.70 160 K160L 0.95 160 K160I 1.00 161 T161R 0.01 161 T161H 0.01 161 T161W 0.01 161 T161N 0.01 161 T161G 0.43 161 T161C 0.56 161 T161S 0.57 161 T161I 0.98 163 E163F 0.27 163 E163R 0.49 163 E163V 0.55 163 E163P 0.77 163 E163G 0.80 163 E163H 0.82 163 E163S 0.85 163 E163W 0.98 164 L164Y 0.01 164 L164A 0.01 164 L164D 0.01 164 L164E 0.01 164 L164G 0.01 164 L164H 0.12 164 L164F 0.86 164 L164C 0.91 164 L164T 0.99 165 A165I 0.59 165 A165K 0.82 165 A165Y 0.84 165 A165S 0.94 165 A165F 1.00 166 R166T 0.74 166 R166V 0.76 166 R166G 0.91 166 R166S 0.95 168 Y168G 0.01 168 Y168T 0.01 168 Y168V 0.01 168 Y168I 0.01 168 Y168C 0.01 168 Y168Q 0.01 169 S169P 0.89 169 S169T 0.97 170 A170I 0.44 170 A170S 0.47 170 A170G 0.62 170 A170T 0.72 170 A170V 0.74 170 A170K 0.83 170 A170W 0.83 170 A170L 0.85 170 A170Q 0.89 170 A170Y 0.89 171 L171R 0.01 172 A172K 0.01 172 A172R 0.01 172 A172E 0.01 172 A172Q 0.18 172 A172V 0.39 172 A172W 0.45 172 A172P 0.58 172 A172I 0.58 172 A172T 0.71 172 A172N 0.76 172 A172G 0.84 172 A172S 0.85 172 A172C 0.86 174 F174W 0.01 174 F174Q 0.46 174 F174C 0.48 174 F174R 0.52 174 F174S 0.61 174 F174T 0.64 174 F174V 0.67 174 F174G 0.91 175 M175P 0.08 175 M175A 0.66 175 M175Y 0.72 175 M175G 0.75 175 M175W 0.76 175 M175V 0.81 175 M175Q 0.83 175 M175L 0.86 175 M175R 0.86 175 M175T 0.90 176 K176S 0.72 176 K176G 0.73 176 K176P 0.78 176 K176L 0.92 176 K176Y 0.93 176 K176N 0.94 176 K176T 0.97 176 K176Q 0.97 178 P178W 0.02 179 F179Q 0.01 179 F179S 0.34 179 F179W 0.86 179 F179H 0.93 179 F179N 0.95 180 F180K 0.01 180 F180T 0.01 180 F180R 0.01 180 F180S 0.01 180 F180G 0.01 180 F180Q 0.01 181 D181Y 0.01 181 D181W 0.01 181 D181L 0.01 181 D181T 0.01 181 D181V 0.01 181 D181R 0.22 181 D181K 0.47 181 D181G 0.52 181 D181S 0.55 181 D181Q 0.60 181 D181P 0.66 181 D181E 0.72 181 D181C 0.85 182 A182I 0.01 182 A182R 0.01 182 A182Q 0.01 182 A182P 0.01 182 A182T 0.11 182 A182N 0.53 182 A182S 0.85 182 A182G 0.94 182 A182C 0.99 183 G183S 0.01 183 G183Q 0.01 183 G183V 0.01 183 G183F 0.19 183 G183H 0.95 183 G183D 0.99 184 S184T 0.60 184 S184H 0.74 184 S184G 0.82 184 S184P 0.85 185 V185W 0.01 185 V185H 0.01 185 V185G 0.01 185 V185D 0.01 185 V185S 0.53 185 V185Y 0.58 185 V185I 0.63 185 V185R 0.79 185 V185K 0.79 185 V185C 0.83 185 V185E 0.88 185 V185T 0.91 185 V185L 0.93 186 I186G 0.01 186 I186S 0.01 186 I186R 0.01 186 I186P 0.01 186 I186T 0.23 186 I186V 0.48 186 I186F 0.76 187 S187P 0.01 187 S187T 0.23 187 S187Q 0.35 187 S187W 0.52 187 S187R 0.55 187 S187V 0.58 187 S187F 0.65 187 S187Y 0.80 188 T188H 0.01 188 T188R 0.01 188 T188F 0.01 188 T188Y 0.09 188 T188I 0.10 188 T188V 0.15 188 T188L 0.42 188 T188M 0.75 188 T188G 0.79 188 T188C 0.87 188 T188S 0.91 188 T188A 0.95 189 D189F 0.37 189 D189R 0.39 189 D189N 0.57 189 D189V 0.71 189 D189W 0.76 189 D189E 0.77 189 D189G 0.80 189 D189S 0.81 189 D189M 0.88 189 D189C 0.94 189 D189H 0.95 189 D189P 0.97 190 G190V 0.01 190 G190S 0.01 190 G190Q 0.29 190 G190W 0.41 190 G190R 0.51 190 G190K 0.57 190 G190L 0.82 191 V191H 0.01 191 V191W 0.01 191 V191S 0.01 191 V191G 0.01 191 V191N 0.01 191 V191I 0.02 192 D192S 0.01 192 D192P 0.01 192 D192F 0.01 192 D192H 0.01 192 D192I 0.01 192 D192Q 0.01 192 D192R 0.01 192 D192T 0.01 192 D192V 0.01 192 D192W 0.01 192 D192N 0.15 192 D192C 0.56 193 G193H 0.01 193 G193C 0.01 193 G193T 0.01 193 G193N 0.01 194 I194S 0.01 194 I194A 0.01 194 I194C 0.01 194 I194P 0.01 194 I194F 0.01 194 I194W 0.01 194 I194R 0.01 194 I194Y 0.01 194 I194G 0.04 194 I194L 0.58 194 I194V 0.78 195 H195S 0.08 195 H195C 0.10 195 H195L 0.18 195 H195N 0.22 195 H195R 0.24 195 H195F 0.40 195 H195V 0.60 195 H195Q 0.96 195 H195A 0.98 196 F196H 0.01 196 F196G 0.01 196 F196S 0.01 196 F196Q 0.01 196 F196W 0.38 196 F196P 0.39 196 F196V 0.68 196 F196M 0.71 196 F196Y 0.97 197 T197R 0.01 197 T197L 0.65 197 T197S 0.75 197 T197G 0.81 197 T197I 0.84 197 T197C 0.86 197 T197V 0.89 197 T197N 0.91 199 A199M 0.93 199 A199S 0.99 199 A199G 0.99 201 N201Y 0.01 201 N201T 0.01 201 N201V 0.01 201 N201R 0.01 201 N201S 0.06 201 N201H 0.10 201 N201G 0.30 201 N201L 0.35 201 N201F 0.67 201 N201E 0.72 203 D203V 0.50 203 D203W 0.52 203 D203E 0.90

The following Table provides variants that have protein performance indices (“Prot. PI”) better than wild-type.

TABLE 10-8 Sites with Protein PI Values Better Than Wild-Type Pos WT/Pos./Var. Prot. PI 2 A002Y 1.61 2 A002N 1.30 2 A002I 1.25 2 A002V 1.18 2 A002T 1.17 2 A002S 1.15 5 I005M 1.29 7 C007A 1.22 7 C007G 1.07 7 C007M 1.03 8 F008N 1.23 8 F008M 1.05 8 F008G 1.03 8 F008P 1.01 11 S011H 1.06 11 S011A 1.04 11 S011D 1.03 11 S011E 1.01 11 S011Q 1.01 12 L012N 1.06 12 L012Q 1.05 13 T013V 1.17 14 W014Y 1.02 16 W016Y 1.02 17 V017A 1.21 17 V017E 1.11 17 V017F 1.09 17 V017I 1.08 17 V017K 1.06 17 V017T 1.03 18 P018C 2.56 18 P018H 2.50 18 P018L 2.50 18 P018E 2.47 18 P018G 2.47 18 P018N 2.35 18 P018V 2.30 18 P018Q 2.13 18 P018R 2.01 18 P018Y 1.68 18 P018S 1.05 19 V019G 1.39 19 V019A 1.23 19 V019E 1.10 19 V019Q 1.07 19 V019K 1.03 19 V019M 1.00 20 E020G 1.11 20 E020P 1.08 20 E020A 1.08 20 E020N 1.01 20 E020V 1.01 22 G022A 1.07 22 G022I 1.03 23 A023F 1.03 24 P024T 1.43 24 P024G 1.34 24 P024S 1.31 24 P024H 1.15 24 P024I 1.11 24 P024L 1.06 25 T025C 1.37 25 T025V 1.30 25 T025G 1.27 25 T025A 1.23 25 T025I 1.19 25 T025P 1.10 25 T025M 1.04 29 A029G 1.22 29 A029P 1.07 29 A029M 1.06 29 A029D 1.06 29 A029V 1.05 29 A029S 1.05 29 A029T 1.02 29 A029E 1.02 30 P030E 1.20 30 P030A 1.15 30 P030S 1.12 30 P030L 1.07 30 P030Q 1.06 30 P030K 1.06 30 P030H 1.05 30 P030Y 1.04 32 V032M 1.11 32 V032A 1.10 32 V032I 1.08 32 V032Q 1.03 32 V032L 1.01 35 T035C 1.16 36 G036C 1.09 36 G036N 1.08 36 G036Q 1.07 36 G036S 1.06 36 G036A 1.00 37 V037N 1.09 39 A039V 1.18 39 A039E 1.03 46 F046A 1.05 46 F046C 1.01 47 E047I 1.02 54 S054A 1.33 54 S054C 1.21 54 S054E 1.16 54 S054D 1.08 54 S054H 1.06 54 S054N 1.01 54 S054M 1.01 55 A055N 1.12 55 A055S 1.08 56 R056Q 1.02 58 T058V 1.13 60 I060A 1.20 60 I060M 1.14 60 I060V 1.06 60 I060L 1.02 61 D061A 1.41 61 D061N 1.12 61 D061V 1.10 61 D061Y 1.03 61 D061Q 1.02 61 D061L 1.00 62 D062A 1.06 62 D062M 1.06 63 P063S 1.17 63 P063Y 1.12 63 P063M 1.09 63 P063Q 1.08 63 P063A 1.06 63 P063V 1.06 63 P063R 1.02 63 P063T 1.02 64 T064Q 1.13 64 T064M 1.07 64 T064R 1.05 64 T064C 1.05 64 T064S 1.03 66 P066Q 1.91 66 P066G 1.78 66 P066N 1.62 66 P066C 1.51 66 P066I 1.51 66 P066R 1.26 66 P066H 1.23 66 P066V 1.12 66 P066Y 1.08 66 P066A 1.03 66 P066F 1.02 67 R067Q 1.60 67 R067L 1.46 67 R067A 1.39 67 R067V 1.24 67 R067P 1.04 67 R067F 1.01 68 L068A 1.07 68 L068V 1.01 68 L068G 1.00 69 N069C 1.18 69 N069G 1.06 69 N069D 1.05 69 N069S 1.03 70 G070A 1.08 72 S072L 1.07 72 S072A 1.06 72 S072Y 1.03 73 Y073N 1.25 73 Y073Q 1.20 73 Y073C 1.18 73 Y073D 1.09 73 Y073V 1.08 73 Y073M 1.05 73 Y073L 1.03 74 L074I 1.45 74 L074Y 1.19 74 L074V 1.18 74 L074A 1.01 75 P075M 1.22 75 P075S 1.18 75 P075T 1.10 75 P075Y 1.08 75 P075C 1.06 75 P075Q 1.04 75 P075L 1.02 75 P075E 1.00 76 S076W 1.06 77 C077L 1.44 77 C077V 1.33 77 C077A 1.20 77 C077S 1.19 77 C077T 1.18 78 L078I 1.06 78 L078V 1.04 79 A079C 1.16 79 A079E 1.12 79 A079S 1.09 79 A079Q 1.05 79 A079M 1.04 79 A079R 1.02 80 T080S 1.12 80 T080E 1.02 80 T080Q 1.02 82 L082G 1.24 82 L082R 1.15 82 L082V 1.14 82 L082S 1.13 82 L082P 1.11 82 L082M 1.07 82 L082K 1.03 82 L082A 1.00 83 P083G 1.01 84 L084V 1.23 86 L086Q 3.66 89 I089V 1.09 89 I089L 1.07 93 T093Q 2.03 96 T096A 1.32 96 T096V 1.12 96 T096S 1.05 96 T096G 1.03 97 K097A 1.11 97 K097R 1.02 98 A098S 1.17 98 A098T 1.03 98 A098N 1.01 99 Y099S 1.45 99 Y099L 1.39 99 Y099H 1.30 99 Y099A 1.29 99 Y099V 1.28 99 Y099G 1.23 99 Y099W 1.20 99 Y099I 1.11 100 F100M 1.20 100 F100N 1.12 100 F100W 1.06 100 F100S 1.02 101 R101L 1.33 101 R101N 1.11 101 R101Q 1.03 101 R101D 1.02 102 R102Q 1.09 103 T103G 1.20 103 T103S 1.14 103 T103H 1.14 103 T103N 1.07 103 T103K 1.05 103 T103P 1.01 104 P104S 1.44 104 P104V 1.40 104 P104E 1.37 104 P104C 1.34 104 P104N 1.32 104 P104T 1.29 104 P104G 1.25 104 P104Q 1.24 104 P104H 1.11 104 P104I 1.07 104 P104M 1.01 105 L105Y 1.18 105 L105H 1.07 105 L105G 1.07 105 L105C 1.05 105 L105Q 1.03 105 L105T 1.00 105 L105P 1.00 106 D106E 1.02 107 I107S 1.05 107 I107V 1.04 107 I107C 1.00 108 A108G 1.15 108 A108S 1.14 108 A108T 1.08 109 L109E 1.24 109 L1091 1.21 109 L109D 1.15 109 L109N 1.13 109 L109F 1.11 109 L109Q 1.08 109 L109A 1.07 109 L109H 1.06 109 L109V 1.06 109 L109M 1.00 110 G110S 1.01 112 S112N 1.09 112 S112E 1.05 113 V113C 1.06 113 V113N 1.01 114 L114C 1.10 114 L114A 1.03 114 L114M 1.00 115 V115I 1.14 115 V115C 1.14 115 V115A 1.11 115 V115M 1.05 115 V115L 1.02 116 T116N 1.68 116 T116H 1.48 116 T116G 1.44 116 T116C 1.30 116 T116E 1.29 116 T116Q 1.29 116 T116M 1.28 116 T116S 1.24 116 T116Y 1.09 116 T116A 1.08 116 T116R 1.03 116 T116L 1.03 117 Q117S 1.13 117 Q117H 1.12 117 Q117E 1.10 117 Q117T 1.06 117 Q117A 1.03 118 V118C 1.28 118 V118A 1.20 118 V118I 1.01 119 L119C 1.18 119 L119A 1.18 119 L119N 1.14 119 L119I 1.06 119 L119S 1.05 119 L119V 1.04 119 L119E 1.04 119 L119R 1.00 120 T120S 1.35 120 T120E 1.19 120 T120C 1.14 120 T120K 1.12 120 T120N 1.10 120 T120A 1.09 120 T120H 1.07 120 T120Q 1.05 120 T120Y 1.01 120 T120L 1.00 121 S121N 1.17 121 S121L 1.12 121 S121A 1.10 121 S121C 1.09 121 S121G 1.07 121 S121R 1.06 121 S121K 1.04 121 S121E 1.01 121 S121Q 1.01 122 A122N 1.11 122 A122L 1.07 122 A122P 1.07 122 A122M 1.06 122 A122V 1.05 122 A122S 1.05 122 A122E 1.04 122 A122I 1.04 122 A122Q 1.02 124 G124M 1.36 124 G124A 1.20 124 G124N 1.18 124 G124C 1.07 124 G124Q 1.02 125 V125I 1.05 126 G126N 1.04 126 G126E 1.02 126 G126A 1.02 127 T127A 1.10 127 T127S 1.08 127 T127V 1.06 127 T127C 1.04 127 T127G 1.04 127 T127D 1.03 127 T127E 1.03 127 T127M 1.02 128 T128N 1.29 128 T128M 1.28 128 T128Q 1.24 128 T128A 1.23 128 T128H 1.19 128 T128P 1.18 128 T128D 1.14 128 T128K 1.10 128 T128S 1.07 128 T128V 1.05 128 T128R 1.03 128 T128F 1.01 129 Y129F 1.44 129 Y129C 1.42 129 Y129A 1.39 129 Y129D 1.35 129 Y129M 1.28 129 Y129N 1.24 129 Y129L 1.22 129 Y129P 1.11 129 Y129G 1.10 129 Y129S 1.08 129 Y129W 1.01 129 Y129V 1.00 130 P130G 1.11 130 P130E 1.08 130 P130K 1.05 130 P130A 1.03 130 P130M 1.03 133 K133Q 1.13 133 K133S 1.02 133 K133A 1.01 133 K133R 1.01 133 K133E 1.01 135 L135M 1.01 136 V136L 1.03 138 S138A 1.44 138 S138C 1.17 138 S138G 1.09 141 P141A 1.13 141 P141G 1.02 142 L142I 1.05 143 A143G 1.17 145 M145I 1.16 145 M145L 1.07 147 H147L 1.09 147 H147C 1.04 149 W149G 1.39 149 W149A 1.35 149 W149M 1.32 149 W149S 1.28 149 W149F 1.27 149 W149Y 1.15 149 W149Q 1.10 149 W149L 1.06 150 F150A 1.70 150 F150M 1.69 150 F150N 1.52 150 F150C 1.41 150 F150P 1.38 150 F150K 1.33 150 F150E 1.32 150 F150T 1.27 150 F150V 1.26 150 F150W 1.26 150 F150Y 1.24 150 F150I 1.19 150 F150L 1.14 150 F150G 1.13 150 F150H 1.09 151 Q151K 1.04 153 I153N 1.04 157 G157A 1.00 159 Q159E 1.14 159 Q159A 1.13 159 Q159G 1.03 161 T161C 1.01 162 T162C 1.17 162 T162I 1.16 162 T162H 1.08 162 T162L 1.05 162 T162F 1.05 162 T162Y 1.03 164 L164M 1.09 164 L164V 1.08 165 A165G 1.14 165 A165Q 1.05 165 A165S 1.05 166 R166M 1.26 166 R166K 1.19 166 R166G 1.19 166 R166N 1.16 166 R166D 1.16 166 R166A 1.12 166 R166L 1.08 166 R166T 1.04 167 V167L 1.13 167 V167H 1.12 167 V167G 1.08 167 V167M 1.04 167 V167I 1.04 167 V167S 1.04 167 V167C 1.01 168 Y168F 1.28 168 Y168L 1.27 170 A170C 1.02 171 L171I 1.16 172 A172C 1.09 172 A172G 1.07 175 M175Y 1.35 175 M175L 1.19 175 M175W 1.14 175 M175N 1.11 175 M175R 1.02 176 K176R 1.06 176 K176Q 1.02 178 P178E 1.05 182 A182C 1.03 183 G183S 1.08 184 S184E 1.39 184 S184A 1.31 184 S184M 1.25 184 S184G 1.15 184 S184D 1.15 184 S184C 1.14 184 S184Q 1.09 184 S184H 1.07 184 S184N 1.03 184 S184V 1.03 184 S184K 1.02 185 V185I 1.03 186 I186M 1.11 188 T188C 2.04 188 T188I 1.85 188 T188L 1.76 188 T188M 1.60 188 T188V 1.53 188 T188S 1.52 188 T188R 1.41 188 T188A 1.40 188 T188G 1.32 188 T188N 1.24 191 V191C 1.04 194 I194L 1.32 194 I194C 1.17 194 I194A 1.15 194 I194W 1.12 194 I194V 1.03 194 I194Y 1.01 196 F196L 1.09 201 N201H 1.49

The following Table provides variants that have a PAD PI that is greater than 1.5, a PAF that is greater than or equal to 0.1, and a protein PI that is greater than or equal to 0.1

TABLE 10-9 PAD PI > 1.5 with PAF ≧ 0.1 and protein PI ≧ 0.1 Wild-Type Variant Amino Acid/Pos. Amino Acid M1 L K3 A, C, H, I, L R4 A I5 A, C, E, L L6 A C7 K T13 A, C P18 C, E, G, H, L, Q, R, V, Y E20 C, Q D21 A, G, K, L, Y G22 A P24 L E26 L R27 A, K, L F28 D, L P30 T, V D31 L, N V32 A, D, E, G, I, K, L, M, N, Q, W R33 C, G, K, L T35 A, C, I, M G36 K Q40 D, G, K, S, T, W, Y Q41 A, K, L G43 E, L A44 C F46 L V48 A, C, L, M, P I49 A E51 A L53 H N59 A, C, D, E, F, G, K, L, Q, S, T, V, W, Y D61 I, K, R N69 H, I, K, V S72 A, C, G, H, M, N P75 D, G, K, S, T, W, Y S76 D, E, G, M T80 G H81 M P83 A, M D85 F, G L86 C V87 C, L I89 A T96 A, C, L, M A98 D F100 A, M R102 A, L P104 C, E, I, M L105 C, F, W D106 V I107 T G110 E, L V115 G Q117 A, M V118 Q T120 E, I, Y S121 A, C, V T128 F, K, L, R, Y P132 A, C, E, G, L, Q, S, Y K133 L V134 A, M V136 A P140 A P144 H, Y P146 C, F, H, L P148 F Q151 A, C, D, E, H, K, P, R, S, T, Y L152 W I153 F, H, K, P, S, T F154 Y E155 A, L, M, N, P, Y G156 D, M, T G157 H E158 F, K, L, M, N, Y T161 M, Q T162 C, F, G, H, I, K, L, M, N, P, Q, S, W, Y E163 A, L, Y A165 D, L, M R166 A, D, H, L V167 A, C, D, G, H, L, M, P, Q, R, S, T, Y Y168 F, L S169 I L171 A, C, F, K, M, N, Q, S S173 A, C, E, F, I, K, L, M, P, R, V, W, Y F174 A, L, M, Y P178 A, D, E, G, K, L, M, Q, S, T, V, Y F179 L G190 A, H, M V191 A, C, D, E, F, K, L, M, Q, R, Y G193 S, V T197 M E198 C, L, M, N, P, R, W, Y A199 C, K, L, Y R202 A, C, E, F, G, H, I, L, M, S, T, W D203 A, C, H, L, R G205 A V206 C, E, F, G, H, K, L, M, N, P, R A209 E, L E210 D, K Q211 M, N, P S214 A, C, D, F, G, I, K, L, R, T, V, W, L215 E, M, T, V, Y

The following Table provides variants with a PAD PI that is less than 0.5, a PAF that is greater than or equal to 0.1, and a protein PI that is greater than or equal to 0.1.

TABLE 10-10 PAD PI < 0.5 with PAF ≧ 0.1, and Protein PI ≧ 0.1 Wild-Type Amino Acid Residue/Pos. Variant(s) A2 Y R4 I, L, V I5 S L6 S, T, V F8 R D10 G L12 A, C, F, G, K, Q, R, S, T, V W14 F, G, I, K, L, R, S, T, V G15 C, N P18 S V19 M, Q, R G22 K, W A23 G, R, S, T25 G, H, I, K, L, M, P, R, W E26 N, S, T, W R27 P, T, W F28 G A29 T, V T35 N, Q, V G36 S, T L38 G, S Q41 S, V L42 Q, S, T G43 P, Q, S, V D45 R, S, T F46 T E47 P V48 S I49 P, R E50 V E51 I, V G52 H, L, S, V L53 E, G, K, R, S S54 F, G, I, K, L, R, T, V, W, Y A55 I, R, T, V R56 C, G, S, T T57 C, N T58 A, M N59 M, R I60 P D62 C, G, H, I, L, R, S, T, V, W T64 R D65 H, R, S, V, Y P66 G, N, Q R67 E, F, G, L, N, P, Q, T, V, W L68 A, C, E, F, G, H, M, N, P, Q, R, S, T, Y N69 Y G70 C, T S72 W, Y Y73 L, R P75 M, R S76 F, W, Y C77 F, W, Y L78 M A79 C, E, H, M, N, Q, R T80 H, I, K, L, W, Y H81 R, Y L82 G, H, R, S, T, V, W P83 T, V L84 A, T, V, W D85 I, L, V, W L86 H, S, T, V, W V87 A, F, G, S, T, Y I88 T, V I89 S M90 S, T, V L91 T, V T93 S, Y N94 H, L, T, V T96 I, R, W, Y K97 G, I, L, P, Q, S, T, V, Y A98 T Y99 S, V F100 E, K, W R101 K, Q, V, W R102 C, G T103 A, C, F, G, H, I, K, L, N, P, R, S, V, W, Y P104 R, T L105 V I107 P, Q L109 A, D, E, F, H, I, Q, R, S, W G110 Q, S, T M111 G, H, R, S S112 H, R, V, Y L114 Q T116 Y V118 P, R, W L119 C, D, E, F, G, H, I, N, R, S, T, V, W T120 H S121 P A122 D, E, F, G, H, K, R, S G123 C G124 A, H, I, M, Q, R, T, V, W V125 E, R, W G126 I, V, Y T127 E, I, L, Q Y129 A, D, G, K, L, M, R, T, V, W P130 A, E, F, G, H, I, L, S, T, V, W A131 D, W, Y P132 F, H, I, T, V K133 A, C, G, H, I, M, T, V L135 F, Q, S, T, V V137 S S138 I P139 S P140 S P141 G, I, Q, R, S, T, V L142 Q, S, V A143 G, P, W M145 E, G, W W149 A, C, F, G, I, M, Q, S, T F150 G, N, P, W E155 F, R, V G156 I G157 R, S, V Q159 A, C, P K160 G T161 G, H, R, W E163 F, R Y168 C, I, V A170 I, S A172 Q, V F174 C, Q, W F179 Q, S G190 S, V, W V191 G, H, I, N, S, W G193 C, H, T I194 A, C, G, S F196 G, Q, W T197 R N201 G, H, L, R, S, T, V, Y D203 V L208 Q, S, V, Y V212 G L215 A, C, G, K, P, R L216 G, I, T

In addition to the assay results described above, various mutations were found to result in unstable protein such that perhydrolase protein was not expressed. Thus, in contrast to the substitutions that resulted in enhanced expression as compared to wild-type, there were some substitutions that are not as favorable, at least under the conditions used herein. However, it is not intended that the present invention exclude these substitutions, as it is contemplated that these substitutions, taken alone or in combination will find use in alternative embodiments of the present invention.

TABLE 10-11 Mutations that Produced Unstable Protein Wild-Type/ Variant Pos. Amino Acid M1 A, E, F, G, K, N, P, R, S, T, W I5 W C7 L, P, T, W G9 A, C, E, K, L, P, Q, R, V T13 F, R, W G15 H, K, L, R, Y P18 A D21 V F28 H, I, R R33 D, E, H, P, W W34 K T35 K, L, P, W, Y G36 P V37 Q, R L38 W A39 F L42 D A44 D, H, P F46 H V48 W E51 P R56 H, K, P, W, Y T57 W T58 E, G, K, P, R, W, Y L74 D, H, P, Q, R, T C77 N, P L78 A, P, R, S A79 V L86 F I88 R, Y I89 D, R L91 H, K, P, R, W, Y G92 A, D, L, M, P, R, T, W, Y T93 P, R, V, W D95 A, D, G, H, K, L, N, Q, R, S, T, V, W, Y K97 D P104 A, L L105 A, M I107 H, W A108 D, F, H, I, N, P, R G110 L L114 F, K, R, W, Y V115 H, K, V134 D, K, R, W, Y V136 R, W V137 D, E, F, P, R, W S138 E, F, H, L, M, Q, R, W, Y P139 L, W, Y P140 D, K, L, M L142 D, G, M, N, R, T H147 G F154 E, L, P, T161 D, E, P Y168 D, E, H, K, N, P, R, S, W L171 D F179 A, P, R F180 E D181 F, H, I, M, N A182 H, K, L, M, W, Y I186 K, W, Y T188 D, K, P, Q, W F196 A, K, N, R

The following Table provides performance indices obtained in PAF and PAD assays for various variants, as well as the protein performance index.

TABLE 10-12 Performance Indices Wild-Type Res./Pos. Mut. PAF PI PAD PI Prot. PI M1 A −0.12 −0.12 −0.01 M1 E −0.12 −0.12 −0.01 M1 F −0.12 −0.12 −0.01 M1 G −0.12 −0.12 −0.01 M1 I 0.96 1.19 0.31 M1 K −0.12 −0.12 −0.01 M1 L 0.75 2.11 0.30 M1 M 1.00 1.00 1.00 M1 N −0.12 −0.12 −0.01 M1 P −0.12 −0.12 −0.01 M1 R −0.12 −0.12 −0.01 M1 S −0.12 −0.12 −0.01 M1 T −0.12 −0.12 −0.01 M1 V 0.87 0.94 0.52 M1 W −0.12 −0.12 −0.01 A2 A 1.00 1.00 1.00 A2 D 1.30 1.05 0.77 A2 E 0.61 1.38 0.52 A2 F 1.24 0.93 0.89 A2 G 1.15 0.84 0.95 A2 I 1.18 0.61 1.25 A2 N 0.93 0.59 1.30 A2 P 0.52 1.17 0.68 A2 Q 0.81 1.29 0.65 A2 R 0.90 1.17 0.70 A2 S 1.01 0.66 1.15 A2 T 0.98 0.61 1.17 A2 V 0.89 0.60 1.18 A2 W 1.75 1.17 0.53 A2 Y 0.84 0.46 1.61 K3 A 0.86 2.14 0.48 K3 C 0.81 1.52 0.67 K3 E 0.12 3.51 0.11 K3 G 0.72 3.74 0.08 K3 H 1.01 1.89 0.30 K3 I 1.05 2.44 0.16 K3 K 1.00 1.00 1.00 K3 L 1.04 1.84 0.50 K3 M 0.85 1.44 0.71 K3 P 0.80 1.45 0.59 K3 Q 0.87 1.19 0.69 K3 R 0.87 1.29 0.46 K3 S 0.94 1.17 0.44 K3 T 1.01 1.03 0.71 K3 V 0.81 0.84 0.33 K3 Y 1.06 1.39 0.86 R4 A 0.41 1.64 0.29 R4 C 0.71 1.34 0.35 R4 D 0.27 1.18 0.32 R4 E 0.32 0.97 0.25 R4 G 0.79 0.79 0.41 R4 H 0.92 0.99 0.59 R4 I 0.24 0.15 0.18 R4 L 0.21 −0.03 0.18 R4 P 0.14 1.44 0.13 R4 Q 1.03 0.99 0.70 R4 R 1.00 1.00 1.00 R4 S 0.65 0.91 0.64 R4 T 0.80 1.00 0.69 R4 V 0.29 0.08 0.22 R4 W 0.04 0.48 0.12 R4 Y 0.63 0.98 0.39 I5 A 0.60 1.88 0.62 I5 C 0.44 2.47 0.54 I5 D −0.13 3.11 0.06 I5 E 0.67 1.59 0.33 I5 F −0.13 0.15 0.06 I5 G 0.05 −3.88 0.10 I5 H 0.55 0.63 0.18 I5 I 1.00 1.00 1.00 I5 L 0.80 1.63 0.96 I5 M 0.63 1.09 1.29 I5 N −0.13 −2.15 0.12 I5 P −0.13 −0.86 0.08 I5 R −0.13 −6.48 0.08 I5 S 1.02 0.37 0.39 I5 T 1.12 0.72 0.25 I5 V 0.94 0.92 0.54 I5 W −0.13 −0.44 −0.01 L6 A 0.87 1.99 0.26 L6 C 0.85 1.22 0.55 L6 E −0.20 −0.59 0.09 L6 G 0.23 −3.45 0.12 L6 H 0.23 −1.08 0.09 L6 I 1.07 0.82 0.86 L6 K 0.41 −1.16 0.05 L6 L 1.00 1.00 1.00 L6 M 0.92 1.44 0.63 L6 Q −0.20 −1.63 0.12 L6 R 0.06 −1.59 0.12 L6 S 0.58 −1.26 0.23 L6 T 1.06 0.35 0.40 L6 V 1.07 0.35 0.44 L6 W 0.06 −2.97 0.09 C7 A 1.42 1.03 1.22 C7 C 1.00 1.00 1.00 C7 E −0.26 1.63 0.20 C7 G 1.39 0.69 1.07 C7 H 1.73 1.37 0.41 C7 I 1.76 1.48 0.31 C7 K 2.69 2.95 0.21 C7 L −0.26 −0.16 −0.01 C7 M 1.13 0.68 1.03 C7 P −0.26 −0.16 −0.01 C7 R 0.22 −1.04 0.15 C7 S 0.62 −2.83 0.10 C7 T −0.26 −0.16 −0.01 C7 W −0.26 −0.16 −0.01 C7 Y 2.09 0.54 0.67 F8 A 0.55 1.33 0.96 F8 C −0.11 4.01 0.10 F8 F 1.00 1.00 1.00 F8 G 1.09 0.65 1.03 F8 H 1.02 0.64 0.97 F8 K 0.81 0.83 0.95 F8 L 0.77 1.31 0.90 F8 M 0.56 1.11 1.05 F8 N −0.11 0.96 1.23 F8 P 1.00 0.83 1.01 F8 R 1.43 0.46 0.73 F8 S 0.71 −2.75 0.13 F8 T 0.88 0.77 0.94 F8 V 1.18 0.85 0.88 F8 Y 0.96 0.90 0.85 G9 A −0.15 −0.18 −0.01 G9 C −0.15 −0.18 −0.01 G9 E −0.15 −0.18 −0.01 G9 G 1.00 1.00 1.00 G9 H 0.29 −0.06 0.16 G9 K −0.15 −0.18 −0.01 G9 L −0.15 −0.18 −0.01 G9 P −0.15 −0.18 −0.01 G9 Q −0.15 −0.18 −0.01 G9 R −0.15 −0.18 −0.01 G9 T 0.21 −2.56 0.12 G9 V −0.15 −0.18 −0.01 D10 A −0.29 −14.24 0.02 D10 D 1.00 1.00 1.00 D10 E 0.01 0.15 0.72 D10 G 0.41 −0.92 0.17 D10 I 1.28 −6.86 0.04 D10 K 2.13 −5.30 0.02 D10 L 3.97 2.04 0.02 D10 M −0.29 −5.94 0.04 D10 N −0.29 −2.23 0.07 D10 P −0.29 −4.16 0.05 D10 R 0.22 −4.36 0.06 D10 S 0.79 −0.58 0.06 D10 T 1.47 −0.45 0.06 D10 V 0.98 −4.22 0.06 D10 W 3.18 −3.70 0.02 D10 Y 1.51 −4.97 0.03 S11 A 0.25 0.53 1.04 S11 D −0.25 −0.22 1.03 S11 E −0.25 −0.23 1.01 S11 F −0.25 −0.13 0.68 S11 G −0.25 −0.09 0.86 S11 H −0.25 0.33 1.06 S11 I −0.25 0.56 0.63 S11 K −0.25 0.40 0.62 S11 L −0.25 −0.22 0.68 S11 Q −0.25 −0.26 1.01 S11 R −0.25 −0.08 0.69 S11 S 1.00 1.00 1.00 S11 T 0.04 −0.36 0.87 S11 V 0.03 −0.15 0.59 L12 A 1.10 0.07 0.71 L12 C 2.29 0.22 0.81 L12 D 0.04 0.00 0.39 L12 F 0.13 0.17 0.60 L12 G 0.44 −0.06 0.60 L12 H 0.02 0.16 0.77 L12 K 0.18 0.13 0.40 L12 L 1.00 1.00 1.00 L12 N 0.53 0.66 1.06 L12 P 0.03 −0.16 0.31 L12 Q 2.65 0.22 1.05 L12 R 0.23 −0.02 0.34 L12 S 0.54 −0.07 0.80 L12 T 0.68 0.06 0.89 L12 V 0.98 −0.05 0.51 L12 W 0.03 0.02 0.33 T13 A 0.25 1.88 0.72 T13 C 0.56 1.55 0.78 T13 E −0.10 1.09 0.44 T13 F −0.10 −0.11 −0.02 T13 G 0.32 0.77 0.57 T13 I 0.12 1.05 0.69 T13 L 0.55 1.47 0.76 T13 M 0.17 1.47 0.94 T13 N −0.10 2.61 0.27 T13 P −0.10 2.73 0.17 T13 Q 0.01 0.51 0.98 T13 R −0.10 −0.11 −0.02 T13 S 0.73 0.68 0.88 T13 T 1.00 1.00 1.00 T13 V 0.19 0.63 1.17 T13 W −0.10 −0.11 −0.02 W14 A −0.23 0.27 0.94 W14 E 0.06 0.15 0.80 W14 F 0.29 0.22 0.71 W14 G 0.30 −0.97 0.70 W14 I 0.33 −0.42 0.66 W14 K 0.29 −0.17 0.71 W14 L 0.25 −0.36 0.82 W14 N −0.23 −0.12 0.81 W14 P −0.23 −0.29 0.34 W14 R 0.23 −0.40 0.66 W14 S 0.31 −0.99 0.69 W14 T 0.24 −0.77 0.64 W14 V 0.26 −0.49 0.58 W14 W 1.00 1.00 1.00 W14 Y 0.31 0.66 1.02 G15 A 1.54 0.61 0.87 G15 C 0.71 −0.27 0.66 G15 D −0.18 0.01 0.26 G15 E −0.18 −1.42 0.11 G15 G 1.00 1.00 1.00 G15 H −0.18 −0.14 −0.01 G15 K −0.18 −0.14 −0.01 G15 L −0.18 −0.14 −0.01 G15 N 0.46 −0.63 0.71 G15 P −0.18 −5.42 0.09 G15 R −0.18 −0.14 −0.01 G15 S 1.05 0.63 0.76 G15 Y −0.18 −0.14 −0.01 W16 A 0.12 0.55 0.50 W16 D 0.02 0.57 0.32 W16 E 0.06 0.65 0.46 W16 G 0.05 −0.07 0.38 W16 H 0.03 −0.02 0.55 W16 I 0.02 1.06 0.74 W16 K 0.01 1.03 0.73 W16 L −0.48 1.16 0.76 W16 M 0.04 0.37 0.56 W16 N 0.02 −0.03 0.43 W16 P 0.03 0.15 0.37 W16 Q 0.05 0.31 0.47 W16 R 0.03 −0.41 0.30 W16 S 0.09 −0.17 0.39 W16 T 0.03 −0.31 0.41 W16 V 0.01 0.88 0.76 W16 W 1.00 1.00 1.00 W16 Y 0.22 1.09 1.02 V17 A 1.01 0.68 1.21 V17 E 0.82 0.75 1.11 V17 F 0.92 0.85 1.09 V17 G 1.17 0.84 0.93 V17 I 0.95 0.99 1.08 V17 K 0.94 0.84 1.06 V17 L 0.90 1.00 0.76 V17 P 0.77 0.96 0.97 V17 R 1.10 0.94 0.76 V17 S 0.96 1.04 0.89 V17 T 0.93 0.86 1.03 V17 V 1.00 1.00 1.00 V17 Y 0.91 0.88 0.99 P18 A −0.28 −0.94 −0.03 P18 C 1.26 4.16 2.56 P18 E 1.22 4.87 2.47 P18 G 1.07 4.96 2.47 P18 H 1.12 6.05 2.50 P18 L 0.93 7.40 2.50 P18 N 1.33 1.42 2.35 P18 P 1.00 1.00 1.00 P18 Q 1.12 3.26 2.13 P18 R 1.16 3.97 2.01 P18 S 0.11 0.07 1.05 P18 V 1.19 4.85 2.30 P18 Y 1.33 4.17 1.68 V19 A 0.61 0.55 1.23 V19 D 0.77 0.79 0.80 V19 E 0.74 0.62 1.10 V19 G 1.32 0.56 1.39 V19 K 0.96 0.97 1.03 V19 L 1.00 0.91 0.90 V19 M 0.33 0.12 1.00 V19 P 0.00 −0.41 0.76 V19 Q 0.93 0.40 1.07 V19 R 1.03 0.34 0.82 V19 S 1.24 0.57 0.80 V19 V 1.00 1.00 1.00 V19 Y 0.94 0.70 0.92 E20 A 1.29 1.28 1.08 E20 C 1.57 1.76 0.71 E20 D 0.87 1.14 0.97 E20 E 1.00 1.00 1.00 E20 G 2.36 0.78 1.11 E20 H 2.17 1.20 0.92 E20 L 2.20 0.73 0.92 E20 N 1.40 1.34 1.01 E20 P 1.00 1.43 1.08 E20 Q 1.27 1.56 0.99 E20 S 2.01 1.18 0.91 E20 T 2.22 1.25 0.94 E20 V 2.11 1.27 1.01 E20 W 2.94 1.30 0.79 D21 A 1.46 1.75 0.84 D21 D 1.00 1.00 1.00 D21 E 0.84 1.39 0.85 D21 F 1.30 1.41 0.81 D21 G 1.37 1.76 0.93 D21 K 1.58 1.80 0.74 D21 L 1.46 1.57 0.82 D21 P 0.81 0.86 0.74 D21 S 1.24 1.11 0.73 D21 V −0.17 −0.12 −0.02 D21 W 1.55 1.44 0.61 D21 Y 1.30 2.01 0.42 G22 A 1.55 1.66 1.07 G22 E 0.15 1.19 0.56 G22 G 1.00 1.00 1.00 G22 I 0.37 1.03 1.03 G22 K 0.23 −0.22 0.78 G22 L 0.38 1.35 0.84 G22 P 0.28 1.36 0.80 G22 Q 0.35 1.44 0.96 G22 R 0.11 0.56 0.73 G22 S 1.02 0.98 0.94 G22 T 1.03 1.16 0.80 G22 V 0.40 0.85 0.89 G22 W 0.25 0.23 0.58 A23 A 1.00 1.00 1.00 A23 F 0.05 0.44 1.03 A23 G 0.45 0.35 0.93 A23 H 0.16 1.04 0.93 A23 L 0.30 1.30 0.75 A23 M 0.85 0.95 0.90 A23 P −0.11 0.73 0.82 A23 Q 0.23 0.73 0.91 A23 R 0.11 0.28 0.80 A23 S 0.69 0.34 0.87 A23 V 0.20 0.60 0.73 A23 W 0.29 0.80 0.71 A23 Y 0.20 0.96 0.73 P24 A 0.54 0.68 0.88 P24 C 0.54 1.04 0.87 P24 G 0.49 0.76 1.34 P24 H 0.42 0.97 1.15 P24 I 0.42 0.85 1.11 P24 K 0.52 1.36 0.71 P24 L 0.58 1.51 1.06 P24 P 1.00 1.00 1.00 P24 Q 0.50 0.65 0.93 P24 R 0.58 0.91 0.85 P24 S 0.53 0.61 1.31 P24 T 0.44 0.66 1.43 T25 A 1.33 0.86 1.23 T25 C 0.67 0.51 1.37 T25 D 0.03 −0.07 0.87 T25 E 0.08 −0.29 0.98 T25 G 1.86 0.43 1.27 T25 H 0.42 −0.02 0.94 T25 I 1.02 0.35 1.19 T25 K 0.36 0.13 0.87 T25 L 0.40 −0.04 0.95 T25 M 0.29 −0.10 1.04 T25 P 0.97 −0.05 1.10 T25 R 0.32 −0.06 0.94 T25 S 1.60 0.58 0.95 T25 T 1.00 1.00 1.00 T25 V 0.91 0.51 1.30 T25 W 0.33 0.14 0.86 E26 A 1.93 1.45 0.79 E26 C 1.40 0.94 0.82 E26 D 0.65 1.39 0.90 E26 E 1.00 1.00 1.00 E26 G 1.28 0.87 0.82 E26 H 1.33 1.19 0.71 E26 K 1.46 1.47 0.77 E26 L 1.30 1.71 0.77 E26 M 2.00 1.10 0.89 E26 N 1.37 0.48 0.88 E26 P 0.43 0.99 0.63 E26 R 1.48 0.81 0.77 E26 S 1.27 0.28 0.92 E26 T 1.44 0.40 0.82 E26 V 1.39 0.97 0.85 E26 W 1.25 0.47 0.68 R27 A 0.45 2.78 0.67 R27 C 0.35 0.58 0.50 R27 E 0.58 0.93 0.46 R27 G 0.42 0.84 0.24 R27 I 0.72 1.41 0.70 R27 K 1.22 1.55 0.69 R27 L 0.48 2.60 0.51 R27 P 0.93 0.48 0.46 R27 R 1.00 1.00 1.00 R27 S 0.53 0.69 0.56 R27 T 0.41 0.01 0.74 R27 V 0.71 0.94 0.85 R27 W 0.21 −0.59 0.33 F28 A 1.27 1.48 0.92 F28 C 0.93 1.21 0.87 F28 D 0.67 2.07 0.40 F28 E 0.51 1.04 0.85 F28 F 1.00 1.00 1.00 F28 G 0.74 −1.53 0.50 F28 H −0.20 −0.19 −0.01 F28 I −0.20 −0.19 −0.01 F28 L 1.09 2.02 0.51 F28 M 1.33 1.37 0.70 F28 P 0.02 0.39 0.42 F28 R −0.20 −0.19 −0.01 F28 S 1.05 0.70 0.82 F28 V 0.86 0.53 0.85 F28 W 1.16 1.17 0.89 F28 Y 0.99 1.36 0.77 A29 A 1.00 1.00 1.00 A29 C 1.08 1.15 0.76 A29 D 0.87 1.00 1.06 A29 E 1.12 0.84 1.02 A29 G 1.60 0.80 1.22 A29 M 0.67 0.77 1.06 A29 P 0.78 0.62 1.07 A29 R 1.76 0.73 0.81 A29 S 1.49 0.55 1.05 A29 T 1.42 0.47 1.02 A29 V 1.80 0.44 1.05 A29 W 1.91 0.74 0.82 A29 Y 1.70 0.59 0.96 P30 A 1.05 0.92 1.15 P30 E 1.01 1.24 1.20 P30 G 0.90 1.09 0.99 P30 H 1.01 1.08 1.05 P30 I 0.97 1.38 0.95 P30 K 1.21 1.39 1.06 P30 L 0.96 1.17 1.07 P30 M 0.96 0.79 0.94 P30 P 1.00 1.00 1.00 P30 Q 1.01 0.91 1.06 P30 R 1.16 1.14 0.94 P30 S 1.03 1.49 1.12 P30 T 1.05 1.64 1.00 P30 V 1.06 1.74 0.99 P30 Y 0.79 1.31 1.04 D31 A 1.24 1.18 0.80 D31 D 1.00 1.00 1.00 D31 E 1.13 0.88 0.93 D31 F 1.44 1.39 0.65 D31 G 1.44 1.16 0.79 D31 L 1.81 1.61 0.65 D31 N 1.34 1.55 0.62 D31 Q 1.07 1.13 0.74 D31 R 1.22 1.49 0.50 D31 S 1.15 1.23 0.55 D31 T 1.45 1.11 0.76 D31 V 1.28 1.08 0.50 D31 W 1.83 1.14 0.60 V32 A 0.43 3.64 1.10 V32 D 0.45 4.19 0.95 V32 E 0.57 3.92 1.00 V32 G 0.58 2.65 0.98 V32 I 0.91 3.51 1.08 V32 K 1.09 4.73 0.75 V32 L 0.96 4.72 1.01 V32 M 0.64 3.41 1.11 V32 N 0.54 1.61 0.99 V32 P 0.01 −1.17 0.31 V32 Q 0.64 1.74 1.03 V32 R 1.05 0.72 0.51 V32 S 0.77 1.09 0.85 V32 V 1.00 1.00 1.00 V32 W 0.94 1.71 0.70 R33 A 0.20 1.32 0.52 R33 C 0.44 1.73 0.95 R33 D −0.16 −0.30 −0.02 R33 E −0.16 −0.30 −0.02 R33 G 0.64 2.63 0.47 R33 H −0.16 −0.30 −0.02 R33 K 0.85 2.72 0.81 R33 L 0.34 2.90 0.74 R33 N 0.90 1.30 0.92 R33 P −0.16 −0.30 −0.02 R33 R 1.00 1.00 1.00 R33 S 1.00 1.01 0.79 R33 V 0.50 0.94 0.89 R33 W −0.16 −0.30 −0.02 W34 A −0.15 2.29 0.41 W34 C −0.15 1.49 0.52 W34 E −0.15 −1.86 0.17 W34 G 0.12 0.88 0.23 W34 I 0.18 0.94 0.75 W34 K −0.15 −0.15 −0.02 W34 M 0.16 1.22 0.91 W34 P −0.15 1.21 0.26 W34 Q 0.02 0.04 0.25 W34 R 0.22 −0.33 0.16 W34 S 0.47 0.08 0.29 W34 T 0.36 0.15 0.29 W34 V 0.24 0.73 0.71 W34 W 1.00 1.00 1.00 T35 A 0.45 3.85 0.98 T35 C 0.55 4.72 1.16 T35 E 0.30 5.73 0.26 T35 I 0.63 5.38 0.45 T35 K −0.13 −0.54 −0.01 T35 L −0.13 −0.54 −0.01 T35 M 0.17 2.72 0.40 T35 N 0.20 −2.29 0.43 T35 P −0.13 −0.54 −0.01 T35 Q 0.57 −2.07 0.52 T35 R 0.18 −11.34 0.23 T35 T 1.00 1.00 1.00 T35 V 0.71 0.34 0.81 T35 W −0.13 −0.54 −0.01 T35 Y −0.13 −0.54 −0.01 G36 A 0.63 1.07 1.00 G36 C 0.53 1.06 1.09 G36 D −0.12 2.50 0.28 G36 G −0.12 −0.10 −0.02 G36 H 0.73 1.10 0.98 G36 I 1.32 1.81 0.31 G36 K 1.27 1.71 0.84 G36 L 1.24 2.49 0.39 G36 M 0.85 0.54 0.85 G36 N 0.49 0.56 1.08 G36 P −0.12 −0.10 −0.02 G36 Q 0.56 0.71 1.07 G36 R 0.99 0.90 0.85 G36 S 0.78 0.26 1.06 G36 T 0.76 0.33 0.83 G36 V 0.95 0.38 0.42 G36 W 0.91 0.68 0.57 V37 A 1.25 2.00 0.63 V37 C 1.09 1.63 0.68 V37 H 1.21 0.96 0.78 V37 I 1.26 1.04 0.77 V37 L 1.16 1.16 0.71 V37 N 0.90 1.52 1.09 V37 P 0.53 2.10 0.73 V37 Q −0.11 −0.14 −0.02 V37 R −0.11 −0.14 −0.02 V37 S 1.40 1.49 0.81 V37 T 1.05 0.81 0.63 V37 V −0.11239 −0.14412 −0.02 V37 W 0.92 0.98 0.62 L38 A 0.59 0.63 0.78 L38 C 0.64 0.72 0.89 L38 D −0.15 0.12 0.24 L38 E −0.15 −0.61 0.26 L38 G 0.15 −0.72 0.32 L38 K 0.63 −0.22 0.16 L38 L 1.00 1.00 1.00 L38 P −0.15 −0.78 0.28 L38 Q −0.15 −0.02 0.47 L38 R −0.15 −0.96 0.34 L38 S 0.38 0.29 0.48 L38 V 0.88 1.12 0.73 L38 W −0.15 −0.11 −0.02 A39 A 1.00 1.00 1.00 A39 C 0.63 0.92 0.50 A39 E 1.09 0.83 1.03 A39 F −0.17 −0.11 −0.02 A39 G 1.17 0.30 0.92 A39 I 1.26 0.71 0.91 A39 K 1.36 0.96 0.90 A39 L 1.43 0.97 0.93 A39 M 0.52 0.81 0.46 A39 N 0.51 0.43 0.45 A39 P 0.69 0.74 0.45 A39 R 1.17 0.64 0.94 A39 S 0.49 −4.31 0.16 A39 T 1.26 0.79 0.92 A39 V 1.21 0.98 1.18 A39 W 1.23 1.02 0.94 A39 Y 1.36 1.13 0.90 Q40 D 1.16 1.59 0.69 Q40 E 1.08 1.28 0.81 Q40 G 1.79 2.17 0.93 Q40 I 2.58 1.10 0.49 Q40 K 2.61 3.64 0.52 Q40 L 2.14 1.49 0.53 Q40 N 1.53 1.00 0.78 Q40 P 0.45 −0.19 0.24 Q40 Q 1.00 1.00 1.00 Q40 R 1.89 1.48 0.61 Q40 S 1.57 1.65 0.87 Q40 T 2.01 1.81 0.75 Q40 W 2.39 2.59 0.54 Q40 Y 1.83 2.02 0.65 Q41 A 1.03 2.58 0.73 Q41 G 0.97 1.09 0.77 Q41 H 1.12 1.14 0.89 Q41 K 1.38 1.61 0.70 Q41 L 1.00 1.92 0.79 Q41 P 0.21 0.66 0.45 Q41 Q 1.00 1.00 1.00 Q41 R 1.19 1.27 0.74 Q41 S 1.11 0.22 0.92 Q41 V 1.07 −0.05 0.90 Q41 W 1.14 0.88 0.71 Q41 Y 1.09 0.70 0.82 L42 C 0.76 1.43 0.68 L42 D −0.14 −0.17 −0.02 L42 F 1.07 1.02 0.48 L42 G 1.17 0.76 0.50 L42 H 1.92 −0.33 0.15 L42 I 0.97 0.66 0.83 L42 K 2.46 1.41 0.13 L42 L 1.00 1.00 1.00 L42 M 0.78 0.74 0.95 L42 P 0.71 1.34 0.23 L42 Q 0.57 0.28 0.40 L42 R 1.38 0.64 0.15 L42 S 0.97 0.45 0.46 L42 T 1.08 −0.04 0.41 L42 V 0.91 0.73 0.74 L42 W 2.06 −0.70 0.14 G43 A 1.49 1.07 0.45 G43 C 1.48 0.73 0.36 G43 E 1.25 1.88 0.66 G43 G 1.00 1.00 1.00 G43 H 1.17 0.96 0.63 G43 I 0.94 0.77 0.42 G43 K 1.42 0.86 0.65 G43 L 1.22 1.82 0.42 G43 M 1.37 0.88 0.28 G43 P 1.08 0.31 0.65 G43 Q 0.91 0.48 0.63 G43 R 1.22 0.59 0.57 G43 S 1.18 0.23 0.79 G43 V 0.93 0.33 0.44 G43 Y 1.26 0.94 0.36 A44 A 1.00 1.00 1.00 A44 C 1.80 1.92 0.46 A44 D −0.17 −0.11 −0.01 A44 E −0.17 0.03 0.10 A44 F 2.84 0.80 0.99 A44 H −0.17 −0.11 −0.01 A44 L 1.61 0.99 0.87 A44 M 1.20 0.98 0.71 A44 P −0.17 −0.11 −0.01 A44 R 0.29 −2.17 0.08 A44 S 0.52 −0.92 0.16 A44 T 0.30 −1.11 0.14 A44 V 2.13 0.50 0.94 A44 W 1.40 0.85 0.61 A44 Y 0.30 −0.23 0.10 D45 A 1.04 0.84 0.99 D45 C 0.83 0.84 0.48 D45 D 1.00 1.00 1.00 D45 F 1.11 1.04 0.66 D45 G 1.13 0.84 0.94 D45 H 1.13 0.78 0.70 D45 K 1.34 0.87 0.86 D45 L 1.05 0.78 0.55 D45 M 0.86 0.78 0.88 D45 P 0.75 0.53 0.72 D45 Q 1.04 0.57 0.81 D45 R 1.16 0.49 0.72 D45 S 1.13 0.38 0.95 D45 T 1.27 0.44 0.86 D45 V 1.05 0.50 0.70 D45 W 1.15 0.58 0.54 F46 A 0.92 1.25 1.05 F46 C 0.84 1.16 1.01 F46 D 1.17 1.39 0.54 F46 E 1.25 1.31 0.38 F46 F 1.00 1.00 1.00 F46 G 1.02 0.94 0.61 F46 H −0.13 −0.13 −0.01 F46 I 0.90 0.88 0.91 F46 K 1.00 1.46 0.48 F46 L 0.78 1.54 0.74 F46 M 0.78 1.42 0.81 F46 P 0.64 1.50 0.26 F46 S 0.73 0.66 0.72 F46 T 0.86 0.43 0.79 F46 V 0.82 0.79 0.89 F46 W 0.94 0.63 0.91 E47 A 0.95 0.76 0.84 E47 C 0.83 0.77 0.99 E47 D 0.99 0.98 0.97 E47 E 1.00 1.00 1.00 E47 F 1.09 0.76 0.96 E47 G 1.20 1.10 0.76 E47 H 1.27 0.99 0.93 E47 I 1.03 1.15 1.02 E47 K 1.19 1.06 0.89 E47 L 1.00 1.02 0.96 E47 M 0.90 0.70 0.84 E47 N 0.91 0.63 0.99 E47 P 1.36 0.36 0.49 E47 R 2.45 0.62 0.75 E47 S 1.28 0.63 0.83 E47 T 1.96 0.84 0.98 V48 A 0.60 1.63 0.47 V48 C 0.83 2.25 0.91 V48 E 0.02 0.99 0.18 V48 F 0.67 1.42 0.57 V48 G 0.61 0.87 0.25 V48 L 0.92 2.29 0.91 V48 M 0.85 1.79 0.71 V48 N −0.15 0.98 0.23 V48 P 0.21 3.08 0.34 V48 Q 0.19 1.39 0.32 V48 R 0.76 −1.17 0.15 V48 S 0.65 0.42 0.40 V48 V 1.00 1.00 1.00 V48 W −0.15 −0.19 −0.02 I49 A 0.92 1.87 0.58 I49 E 1.02 0.88 0.75 I49 G 1.34 1.12 0.28 I49 H 1.27 0.74 0.77 I49 I 1.00 1.00 1.00 I49 K 1.23 1.26 0.72 I49 L 1.14 1.03 0.93 I49 M 1.01 1.02 0.69 I49 P 0.47 0.16 0.29 I49 R 1.05 0.29 0.56 I49 S 1.24 0.79 0.70 I49 V 1.20 0.97 0.94 I49 W 0.70 0.68 0.64 I49 Y 1.07 1.02 0.82 E50 A 1.12 1.23 0.58 E50 D 0.78 1.22 0.80 E50 E 1.00 1.00 1.00 E50 G 0.93 1.11 0.60 E50 I 0.84 0.58 0.67 E50 L 1.19 0.97 0.41 E50 M 1.18 1.04 0.38 E50 P 0.85 1.02 0.71 E50 Q 0.98 0.91 0.70 E50 R 0.46 −0.77 0.20 E50 S 0.87 0.65 0.76 E50 V 1.00 0.43 0.81 E50 W 0.75 0.14 0.19 E51 A 1.28 2.72 0.74 E51 D 0.66 1.28 0.91 E51 E 1.00 1.00 1.00 E51 G 1.22 1.34 0.84 E51 I 1.07 0.04 0.52 E51 K 0.38 2.00 0.36 E51 L 1.11 0.93 0.57 E51 M 0.40 1.20 0.84 E51 P −0.12 −0.39 −0.02 E51 Q 0.98 0.76 0.84 E51 R 0.35 −0.97 0.29 E51 T 1.18 1.17 0.48 E51 V 1.47 0.37 0.70 E51 W 0.44 0.17 0.22 G52 A 0.54 0.79 0.90 G52 E −0.12 0.55 0.41 G52 F −0.12 −0.08 0.52 G52 G 1.00 1.00 1.00 G52 H 0.18 −0.60 0.49 G52 I 0.10 0.07 0.80 G52 L 0.17 0.24 0.58 G52 M 0.05 −0.64 0.56 G52 P −0.12 0.24 0.76 G52 Q −0.12 0.28 0.52 G52 R −0.12 0.35 0.18 G52 S 0.13 −0.18 0.83 G52 T 0.10 −0.17 0.76 G52 V 0.10 −0.16 0.86 G52 W 0.92 2.47 0.13 L53 D 0.01 0.01 0.72 L53 E 0.88 0.19 0.77 L53 G 1.32 0.33 0.80 L53 H 5.05 1.70 0.27 L53 I 0.55 0.66 0.88 L53 K 0.89 0.24 0.70 L53 L 1.00 1.00 1.00 L53 P −0.11 −0.64 0.07 L53 Q 1.48 0.72 0.89 L53 R 0.20 −0.02 0.66 L53 S 1.16 0.26 0.95 L53 T 1.02 0.84 0.75 L53 V 0.52 0.65 0.88 L53 W 0.02 −0.07 0.77 S54 A 3.46 1.41 1.33 S54 C 1.26 0.88 1.21 S54 D −0.17 0.65 1.08 S54 E −0.17 0.30 1.16 S54 F 0.74 −0.14 0.91 S54 G 1.43 0.17 0.93 S54 H −0.17 0.00 1.06 S54 I 4.78 0.12 0.94 S54 K 1.44 0.08 0.78 S54 L 2.02 0.26 0.59 S54 M 0.01 0.48 1.01 S54 N 0.29 1.29 1.01 S54 P 5.20 1.30 0.98 S54 Q 1.03 0.53 0.99 S54 R 3.38 0.35 0.84 S54 S 1.00 1.00 1.00 S54 T 1.46 0.33 0.88 S54 V 4.72 0.29 0.95 S54 W 0.11 −0.07 0.83 S54 Y 0.37 0.12 0.89 A55 A −0.11 −0.15 −0.01 A55 C 0.14 1.26 0.98 A55 G 1.69 0.73 0.98 A55 H 0.04 0.92 0.93 A55 I 0.34 −0.43 0.80 A55 K 0.52 1.08 0.68 A55 L 0.11 0.87 0.81 A55 N 0.34 1.05 1.12 A55 P −0.11 −0.01 0.84 A55 R 0.56 0.25 0.99 A55 S 0.76 0.87 1.08 A55 T 1.69 0.42 0.91 A55 V 0.49 −0.51 0.96 A55 W 0.00 −0.05 0.88 A55 Y 0.00 0.18 0.94 R56 A 0.22 0.69 0.85 R56 C 0.45 −0.02 0.93 R56 E −0.12 −0.04 0.16 R56 G 0.30 −0.59 0.56 R56 H −0.12 −0.37 −0.02 R56 K −0.12 −0.37 −0.02 R56 L 0.05 0.24 0.87 R56 N 0.18 0.27 0.31 R56 P −0.12 −0.37 −0.02 R56 Q 0.01 −0.01 1.02 R56 R 1.00 1.00 1.00 R56 S 0.39 0.12 0.55 R56 T 0.10 −0.37 0.85 R56 W −0.12 −0.37 −0.02 R56 Y −0.12 −0.37 −0.02 T57 A 0.60 0.65 0.59 T57 C 0.60 0.40 0.85 T57 G 0.92 1.05 0.53 T57 H 0.83 0.61 0.23 T57 I 1.19 0.87 0.65 T57 L 0.63 0.76 0.95 T57 N 0.89 0.25 0.69 T57 P 0.33 −0.87 0.13 T57 R 1.61 −0.66 0.14 T57 S 1.63 1.01 0.88 T57 T 1.00 1.00 1.00 T57 V 1.28 0.87 0.84 T57 W −0.08 −0.10 −0.01 T57 Y 0.52 0.55 0.43 T58 A 0.65 0.36 0.76 T58 E −0.19 −0.10 −0.02 T58 G −0.19 −0.10 −0.02 T58 H 0.89 1.49 0.74 T58 K −0.19 −0.10 −0.02 T58 L 0.88 1.12 0.78 T58 M 0.56 0.03 0.50 T58 P −0.19 −0.10 −0.02 T58 R −0.19 −0.10 −0.02 T58 S 0.82 0.96 0.90 T58 T 1.00 1.00 1.00 T58 V 0.56 0.96 1.13 T58 W −0.19 −0.10 −0.02 T58 Y −0.19 −0.10 −0.02 N59 A 0.35 10.44 0.73 N59 C 0.40 11.23 0.78 N59 D 0.52 11.72 0.67 N59 E 0.66 9.88 0.38 N59 F 0.82 10.23 0.57 N59 G 0.88 10.00 0.66 N59 K 0.89 8.21 0.31 N59 L 0.88 14.74 0.32 N59 M 0.42 −1.42 0.72 N59 N 1.00 1.00 1.00 N59 P 0.12 −55.11 0.14 N59 Q 1.02 1.86 0.73 N59 R 1.09 −11.28 0.39 N59 S 1.06 7.32 0.74 N59 T 1.07 5.63 0.56 N59 V 0.81 9.97 0.96 N59 W 1.13 12.80 0.59 N59 Y 0.80 11.14 0.61 I60 A 0.81 0.79 1.20 I60 C 0.69 0.67 0.97 I60 D 0.83 0.66 0.56 I60 E 0.87 0.92 0.83 I60 G 1.00 1.04 0.86 I60 H 1.02 1.07 0.96 I60 I 1.00 1.00 1.00 I60 K 0.99 0.96 0.73 I60 L 0.95 0.91 1.02 I60 M 0.96 0.68 1.14 I60 P 0.23 0.32 0.31 I60 R 1.00 0.81 0.79 I60 S 0.78 1.00 0.92 I60 V 0.87 1.06 1.06 I60 Y 0.78 1.19 0.89 D61 A 0.70 0.71 1.41 D61 C 0.79 0.85 0.92 D61 D 1.00 1.00 1.00 D61 F 1.01 0.70 0.61 D61 G 0.81 1.25 0.84 D61 H 1.44 1.67 0.97 D61 I 1.08 1.66 0.98 D61 K 0.92 1.72 0.97 D61 L 0.80 1.20 1.00 D61 N 0.79 1.00 1.12 D61 P 0.83 1.13 0.97 D61 Q 0.89 1.16 1.02 D61 R 1.11 1.59 0.69 D61 S 1.26 1.35 0.97 D61 V 0.95 0.97 1.10 D61 Y 0.84 0.95 1.03 D62 A −0.24 0.11 1.06 D62 C 0.52 0.49 0.96 D62 E 1.02 0.60 0.93 D62 G 0.28 −0.21 0.86 D62 H 0.61 −0.01 0.89 D62 I 0.72 −0.25 0.92 D62 L 0.51 −0.37 0.95 D62 M 0.03 −0.24 1.06 D62 P −0.24 −0.55 0.69 D62 Q −0.24 −0.35 0.86 D62 R 0.12 −0.81 0.62 D62 S 0.57 −0.10 0.88 D62 T 0.76 −0.41 0.76 D62 V 0.62 −0.26 0.87 D62 W 0.58 −0.45 0.79 P63 A 1.35 0.60 1.06 P63 F 1.25 0.93 0.97 P63 G 1.71 1.22 1.00 P63 K 1.40 1.02 0.99 P63 L 1.15 1.23 0.84 P63 M 1.46 0.91 1.09 P63 Q 1.09 1.05 1.08 P63 R 1.31 0.80 1.02 P63 S 1.42 0.90 1.17 P63 T 1.50 1.32 1.02 P63 V 1.31 1.04 1.06 P63 W 1.35 1.11 0.86 P63 Y 1.35 0.95 1.12 T64 A 0.96 1.20 0.97 T64 C 0.78 0.88 1.05 T64 D 0.87 0.64 0.81 T64 G 1.23 1.08 1.00 T64 H 0.89 0.96 0.90 T64 L 0.63 1.22 0.93 T64 M 0.68 1.09 1.07 T64 N 0.69 0.98 0.91 T64 P 0.76 0.94 0.61 T64 Q 0.76 0.87 1.13 T64 R 0.15 0.11 1.05 T64 S 1.11 0.99 1.03 T64 T 1.00 1.00 1.00 T64 W 0.71 0.69 0.72 D65 A 1.31 0.72 0.72 D65 D 1.00 1.00 1.00 D65 G 0.80 0.52 0.88 D65 H 1.10 0.40 0.71 D65 I 0.53 0.62 0.46 D65 P −0.33 0.42 0.08 D65 R 0.41 0.22 0.84 D65 S 1.17 0.47 0.76 D65 T 0.90 0.50 0.68 D65 V 0.88 0.20 0.64 D65 W 0.77 0.50 0.65 D65 Y 0.83 0.42 0.64 P66 A 0.50 0.56 1.03 P66 C 0.51 0.52 1.51 P66 D 1.00 0.72 0.90 P66 F 0.95 0.67 1.02 P66 G 1.50 0.44 1.78 P66 H 1.59 0.95 1.23 P66 I 1.59 0.84 1.51 P66 L 1.14 0.99 0.92 P66 N 1.12 0.38 1.62 P66 P −0.09 −0.11 −0.01 P66 Q 1.46 0.42 1.91 P66 R 1.85 0.51 1.26 P66 S 1.39 1.02 0.98 P66 T 1.41 1.10 0.72 P66 V 1.83 0.89 1.12 P66 Y 1.33 0.70 1.08 R67 A −0.20 0.22 1.39 R67 E 1.04 0.11 0.85 R67 F 1.26 0.01 1.01 R67 G 1.39 0.41 0.81 R67 K 0.91 0.99 0.76 R67 L 1.20 0.16 1.46 R67 N 1.58 0.33 1.00 R67 P 1.01 0.04 1.04 R67 Q 1.16 0.13 1.60 R67 R 1.00 1.00 1.00 R67 T 1.28 0.32 0.76 R67 V 0.89 0.12 1.24 R67 W 1.07 0.02 0.95 L68 A 0.59 −0.11 1.07 L68 C 0.76 0.06 0.85 L68 D −0.16 0.44 0.55 L68 E 1.44 0.13 0.87 L68 F 0.70 0.25 1.00 L68 G 1.09 −0.08 1.00 L68 H 1.05 0.22 0.89 L68 I 1.13 0.73 0.86 L68 L 1.00 1.00 1.00 L68 M 0.59 0.03 0.99 L68 N 0.51 0.10 0.95 L68 P 0.29 0.35 0.82 L68 Q 0.50 0.25 0.90 L68 R 0.19 0.47 0.75 L68 S 0.99 0.07 0.93 L68 T 1.03 0.32 0.92 L68 V 1.09 0.51 1.01 L68 W 1.21 0.56 0.88 L68 Y 0.71 0.45 0.97 N69 A 0.92 1.13 0.93 N69 C 1.05 1.20 1.18 N69 D 0.90 1.11 1.05 N69 G 1.20 0.98 1.06 N69 H 1.36 1.52 0.73 N69 I 1.47 1.75 0.69 N69 K 1.72 1.59 0.84 N69 L 1.30 1.20 0.36 N69 N 1.00 1.00 1.00 N69 P 1.00 0.59 0.66 N69 Q 1.07 1.14 0.74 N69 R 1.49 0.83 0.84 N69 S 1.21 1.42 1.03 N69 T 1.35 1.43 0.87 N69 V 1.99 1.73 0.87 N69 W 1.05 0.55 0.36 N69 Y 0.88 0.17 0.44 G70 A 0.85 1.41 1.08 G70 C 0.12 −0.90 0.40 G70 E −0.16 0.33 0.28 G70 F 0.00 −0.36 0.21 G70 G 1.00 1.00 1.00 G70 H 0.04 1.90 0.26 G70 I 0.04 0.27 0.33 G70 K 0.03 −0.80 0.26 G70 L 0.03 1.01 0.30 G70 M 0.62 −0.72 0.29 G70 N 0.02 −0.76 0.37 G70 P 0.16 −0.58 0.29 G70 Q 0.02 −0.83 0.36 G70 R 0.08 −1.84 0.25 G70 S 0.69 0.64 0.88 G70 T 0.27 −0.10 0.45 G70 V 0.16 −0.52 0.34 G70 Y 0.08 −0.33 0.38 A71 A 1.00 1.00 1.00 A71 C 1.01 0.99 0.85 A71 D 0.70 0.65 0.68 A71 E 1.45 0.81 0.83 A71 F 1.13 0.99 0.75 A71 G 1.59 0.68 0.85 A71 H 1.70 0.78 0.75 A71 I 1.51 0.79 0.81 A71 K 1.44 1.01 0.76 A71 L 1.23 0.84 0.85 A71 M 0.98 1.11 0.81 A71 N 1.23 0.61 0.77 A71 P −0.14 −0.05 0.46 A71 R 1.40 0.77 0.71 A71 S 1.75 0.69 0.84 A71 T 1.70 0.79 0.83 S72 A 0.55 3.52 1.06 S72 C 0.56 2.18 0.96 S72 D 0.40 0.80 0.90 S72 E 0.61 0.93 0.99 S72 F 0.94 1.15 0.80 S72 G 1.20 1.76 0.87 S72 H 1.21 2.48 0.82 S72 L 1.26 0.70 1.07 S72 M 0.36 2.13 0.94 S72 N 0.42 2.85 0.99 S72 P −0.25 0.56 0.63 S72 Q 0.62 0.66 0.98 S72 R 0.86 0.74 0.87 S72 S 1.00 1.00 1.00 S72 T 1.10 0.97 0.88 S72 V 1.08 0.83 0.90 S72 W 0.98 0.34 0.92 S72 Y 1.07 0.07 1.03 Y73 A −0.10 1.40 0.82 Y73 C −0.10 1.20 1.18 Y73 D 0.13 0.80 1.09 Y73 G 0.71 0.51 0.95 Y73 H 0.67 0.52 0.96 Y73 I 0.82 0.64 0.97 Y73 K 1.07 0.94 0.95 Y73 L 0.98 0.50 1.03 Y73 M −0.10 1.13 1.05 Y73 N 0.56 0.76 1.25 Y73 P 0.64 −0.54 0.42 Y73 Q 1.23 0.87 1.20 Y73 R 1.26 0.26 0.96 Y73 S 1.17 0.68 0.77 Y73 V 0.88 0.74 1.08 Y73 Y −0.10 −0.10 −0.02 L74 A 0.07 2.90 1.01 L74 D −0.18 −0.18 −0.03 L74 F 0.99 1.13 0.58 L74 G 1.95 0.57 0.18 L74 H −0.18 −0.18 −0.03 L74 I 0.86 0.64 1.45 L74 L 1.00 1.00 1.00 L74 M 0.15 1.21 0.79 L74 P −0.18 −0.18 −0.03 L74 Q −0.18 −0.18 −0.03 L74 R −0.18 −0.18 −0.03 L74 S 2.72 −1.52 0.25 L74 T −0.18 −0.18 −0.03 L74 V 0.90 0.61 1.18 L74 W 1.38 0.67 0.50 L74 Y 0.90 0.86 1.19 P75 C 0.54 1.42 1.06 P75 D 0.67 2.09 0.86 P75 E 0.83 1.19 1.00 P75 G 1.16 0.93 0.81 P75 H 1.05 0.86 0.89 P75 I 0.69 0.74 0.78 P75 K 0.60 0.88 0.91 P75 L 0.44 1.19 1.02 P75 M 0.36 0.30 1.22 P75 P 1.00 1.00 1.00 P75 Q 1.21 0.61 1.04 P75 R 1.60 0.46 0.89 P75 S 1.39 0.63 1.18 P75 T 1.28 0.69 1.10 P75 V 0.93 1.39 0.90 P75 W 1.04 1.31 0.84 P75 Y 0.69 1.32 1.08 S76 A 0.38 1.11 0.60 S76 C 0.39 1.06 0.67 S76 D 0.41 1.94 0.49 S76 E 0.47 2.09 0.58 S76 F 0.44 0.46 0.68 S76 G 0.64 2.15 0.69 S76 H 0.85 1.11 0.79 S76 K 0.59 1.53 0.32 S76 L 0.74 4.70 0.27 S76 M 0.49 1.61 0.45 S76 P 1.23 1.20 0.67 S76 Q 0.84 0.90 0.88 S76 S 1.00 1.00 1.00 S76 T 0.75 1.11 0.80 S76 V 0.67 1.35 0.78 S76 W 0.57 −0.25 1.06 S76 Y 0.31 0.18 0.75 C77 A 0.83 0.91 1.20 C77 C 1.00 1.00 1.00 C77 D 0.92 1.05 0.45 C77 F 0.25 −0.61 0.75 C77 G 1.01 0.18 0.53 C77 L 0.98 0.73 1.44 C77 N −0.13 −0.06 −0.04 C77 P −0.13 −0.06 −0.04 C77 R 0.70 −1.02 0.34 C77 S 0.95 0.76 1.19 C77 T 1.12 1.03 1.18 C77 V 1.05 0.80 1.33 C77 W 0.39 −0.24 0.73 C77 Y 0.95 −0.01 0.66 L78 A −0.11 −0.14 −0.01 L78 C 0.92 0.78 0.91 L78 E 3.01 −1.14 0.16 L78 G 4.98 1.38 0.12 L78 H 4.82 1.57 0.25 L78 I 1.43 1.11 1.06 L78 L 1.00 1.00 1.00 L78 M 0.52 0.48 0.75 L78 N 2.68 −0.41 0.22 L78 P −0.11 −0.14 −0.01 L78 Q 1.73 0.52 0.46 L78 R −0.11 −0.14 −0.01 L78 S −0.11 −0.14 −0.01 L78 T 1.87 1.10 0.47 L78 V 1.53 0.83 1.04 L78 Y 1.39 0.81 0.46 A79 A −0.15 −0.13 −0.02 A79 C 0.97 0.03 1.16 A79 E 1.12 0.27 1.12 A79 F −0.15 −2.02 0.17 A79 G 0.92 0.92 0.99 A79 H 1.93 −0.09 0.85 A79 I 1.59 0.67 0.87 A79 L 1.80 0.96 0.88 A79 M 1.50 0.28 1.04 A79 N 1.48 0.28 0.97 A79 P 0.70 0.94 0.81 A79 Q 1.47 0.27 1.05 A79 R 1.47 0.32 1.02 A79 S 0.82 0.78 1.09 A79 T 1.17 0.60 0.90 A79 V −0.15 −0.13 −0.02 A79 W 1.27 0.53 0.46 T80 A 1.00 1.11 0.90 T80 C 1.31 1.15 0.91 T80 E 0.07 −0.16 1.02 T80 G 1.16 1.50 0.81 T80 H 0.21 0.05 0.66 T80 I 0.50 0.15 0.78 T80 K 0.15 −0.32 0.74 T80 L 0.15 −0.11 0.68 T80 N 0.53 0.53 0.97 T80 P −0.11 −0.05 0.55 T80 Q 0.91 1.07 1.02 T80 R 0.08 −0.22 0.78 T80 S 0.96 1.40 1.12 T80 T 1.00 1.00 1.00 T80 V 1.23 1.01 0.93 T80 W 0.23 −0.86 0.46 T80 Y 0.15 0.11 0.69 H81 A 1.15 1.45 0.98 H81 C 1.13 1.09 0.92 H81 F 1.10 0.90 0.87 H81 G 1.17 0.80 0.94 H81 H 1.00 1.00 1.00 H81 K 1.52 0.56 0.31 H81 L 1.23 1.03 0.93 H81 M 0.94 1.54 0.82 H81 N 1.17 1.00 0.82 H81 P −0.10 0.72 0.42 H81 Q 0.85 0.75 1.00 H81 R 0.34 −0.29 0.85 H81 S 1.04 0.69 0.94 H81 V 1.10 0.71 0.89 H81 W 1.13 1.09 0.90 H81 Y 0.77 0.14 0.76 L82 A 0.62 0.98 1.00 L82 G 1.38 0.31 1.24 L82 H 1.33 0.47 0.95 L82 I 1.17 0.51 0.58 L82 K 1.19 0.51 1.03 L82 L 1.00 1.00 1.00 L82 M 0.65 1.06 1.07 L82 P 1.46 0.52 1.11 L82 R 1.34 −0.18 1.15 L82 S 1.15 0.00 1.13 L82 T 1.18 0.38 0.97 L82 V 1.02 0.19 1.14 L82 W 0.27 −0.46 0.93 P83 A 0.36 2.36 0.66 P83 C 0.53 1.01 0.81 P83 D 0.75 0.83 0.92 P83 E 0.84 1.26 0.92 P83 F 0.76 0.99 0.69 P83 G 1.31 0.68 1.01 P83 H 1.27 0.61 0.93 P83 K 1.37 1.16 0.88 P83 L 0.04 0.21 0.19 P83 M 0.58 1.88 0.71 P83 N 0.70 1.10 0.90 P83 P 1.00 1.00 1.00 P83 Q 0.73 0.82 0.95 P83 R 1.19 1.09 0.78 P83 S 1.17 0.79 0.89 P83 T 0.86 −0.02 0.62 P83 V 0.78 0.19 0.72 P83 W 0.98 0.62 0.69 L84 A 0.45 0.45 0.76 L84 D 0.19 0.85 0.48 L84 F 0.72 1.01 0.74 L84 G 0.77 1.01 0.53 L84 H 1.01 0.99 0.66 L84 I 0.90 0.87 0.99 L84 K 1.10 0.79 0.59 L84 L 1.00 1.00 1.00 L84 N 0.54 0.67 0.86 L84 P −0.12 0.43 0.58 L84 Q 0.41 0.52 0.93 L84 R 0.56 0.57 0.71 L84 S 0.75 0.55 0.93 L84 T 0.86 0.44 0.95 L84 V 0.79 0.42 1.23 L84 W 0.36 −0.28 0.91 D85 A 0.79 1.09 0.63 D85 C 0.88 1.50 0.56 D85 D 1.00 1.00 1.00 D85 E 1.12 1.25 0.97 D85 F 1.01 1.98 0.52 D85 G 1.41 1.60 0.69 D85 H 1.55 1.24 0.76 D85 I 0.55 0.10 0.46 D85 L 0.53 0.24 0.52 D85 N 1.54 0.78 0.86 D85 P 0.97 0.54 0.63 D85 Q 3.09 0.99 0.82 D85 R 2.38 1.03 0.66 D85 S 2.28 0.68 0.93 D85 T 1.33 0.71 0.77 D85 V 0.61 0.25 0.65 D85 W 0.87 0.34 0.72 D85 Y 0.98 0.55 0.78 L86 A 1.38 3.32 0.40 L86 C 1.16 2.44 0.85 L86 E 0.06 −0.92 0.46 L86 F −0.15 −0.26 −0.02 L86 G 1.15 0.70 0.83 L86 H 0.88 −0.72 0.57 L86 L 1.00 1.00 1.00 L86 P −0.15 0.99 0.22 L86 Q −0.15 −2.60 3.66 L86 R 0.43 −4.46 0.26 L86 S 0.78 −0.36 0.78 L86 T 0.96 0.28 0.75 L86 V 0.92 0.12 0.93 L86 W 0.67 0.08 0.78 L86 Y 0.85 0.82 0.92 V87 A 0.65 0.17 0.88 V87 C 0.67 2.22 0.93 V87 D −0.09 −2.53 0.32 V87 F 0.60 0.10 0.56 V87 G 0.46 −2.95 0.54 V87 K 0.04 −8.34 0.26 V87 L 0.71 4.30 0.84 V87 M 0.73 0.75 0.86 V87 P 0.07 1.64 0.39 V87 R 0.07 −1.33 0.44 V87 S 0.59 −0.09 0.67 V87 T 0.63 0.15 0.71 V87 V 1.00 1.00 1.00 V87 Y 0.33 −1.24 0.42 I88 G 1.01 −2.63 0.27 I88 H 1.20 −6.25 0.21 I88 I 1.00 1.00 1.00 I88 M 0.24 1.09 0.86 I88 N −0.14 −0.55 0.29 I88 P −0.14 3.51 0.18 I88 Q 0.01 −1.10 0.36 I88 R −0.14 −0.32 −0.02 I88 T 1.03 −0.16 0.52 I88 Y −0.14 −0.32 −0.02 I89 A 0.55 1.83 0.63 I89 D −0.10 −0.14 −0.02 I89 E −0.10 −2.05 0.24 I89 F 0.68 0.75 0.90 I89 G 0.64 −3.84 0.29 I89 H 1.00 −1.01 0.33 I89 I 1.00 1.00 1.00 I89 L 0.87 1.22 1.07 I89 P 0.38 1.91 0.30 I89 Q 0.25 −0.30 0.32 I89 R −0.10 −0.14 −0.02 I89 S 0.71 −1.66 0.49 I89 T 0.94 0.90 0.60 I89 V 0.91 0.82 1.09 I89 W 0.53 −2.63 0.27 M90 A 0.78 1.41 0.67 M90 C 0.79 1.09 0.83 M90 D −0.24 2.88 0.15 M90 E −0.24 1.15 0.29 M90 G 0.57 −1.22 0.33 M90 I 1.13 0.66 0.74 M90 L 1.02 0.98 0.84 M90 M 1.00 1.00 1.00 M90 P −0.24 −0.36 0.28 M90 Q 0.68 0.77 0.71 M90 R −0.24 0.36 0.23 M90 S 1.06 −0.17 0.56 M90 T 1.27 0.15 0.59 M90 V 1.08 0.08 0.62 M90 W 0.79 −4.04 0.21 L91 A 0.57 1.45 0.81 L91 C 0.67 1.27 0.87 L91 D −0.12 1.47 0.12 L91 E −0.12 −0.51 0.13 L91 G 1.21 −0.58 0.17 L91 H −0.12 −0.13 −0.01 L91 I 0.98 1.05 0.89 L91 K −0.12 −0.13 −0.01 L91 L 1.00 1.00 1.00 L91 M 0.28 0.88 0.80 L91 P −0.12 −0.13 −0.01 L91 Q 0.05 −0.14 0.18 L91 R −0.12 −0.13 −0.01 L91 S 0.92 0.43 0.24 L91 T 1.06 −0.11 0.36 L91 V 0.94 0.79 0.72 L91 W −0.12 −0.13 −0.01 L91 Y −0.12 −0.13 −0.01 G92 A −0.10 −0.18 −0.02 G92 C −0.10 2.05 0.18 G92 D −0.10 −0.18 −0.02 G92 E −0.10 −2.31 0.21 G92 F −0.10 −3.24 0.17 G92 G 1.00 1.00 1.00 G92 L −0.10 −0.18 −0.02 G92 M −0.10 −0.18 −0.02 G92 P −0.10 −0.18 −0.02 G92 R −0.10 −0.18 −0.02 G92 S 1.26 −2.96 0.21 G92 T −0.10 −0.18 −0.02 G92 V 1.49 −3.03 0.20 G92 W −0.10 −0.18 −0.02 G92 Y −0.10 −0.18 −0.02 T93 A 1.38 1.05 0.50 T93 C 1.08 0.95 0.64 T93 D −0.18 0.23 0.22 T93 F 3.52 0.54 0.63 T93 P −0.18 −0.19 −0.02 T93 Q −0.18 −6.75 2.03 T93 R −0.18 −0.19 −0.02 T93 S 0.89 0.49 0.89 T93 T 1.00 1.00 1.00 T93 V −0.18 −0.19 −0.02 T93 W −0.18 −0.19 −0.02 T93 Y 5.26 0.03 0.77 N94 A −0.45 0.74 0.96 N94 C 0.01 0.07 0.94 N94 G 0.15 0.53 0.76 N94 H 0.11 −0.94 0.77 N94 L 0.61 −0.18 0.49 N94 M −0.45 0.03 0.94 N94 N 1.00 1.00 1.00 N94 P −0.45 0.79 0.40 N94 R 0.10 −8.20 0.19 N94 S 0.10 0.88 0.84 N94 T 0.25 −1.43 0.66 N94 V 0.15 −0.39 0.65 N94 W 0.10 −1.20 0.69 N94 Y 0.08 0.12 0.76 D95 A −0.14 −0.14 −0.01 D95 C −0.14 −0.14 −0.01 D95 D 1.00 1.00 1.00 D95 E 2.04 0.75 0.66 D95 G −0.14 −0.14 −0.01 D95 H −0.14 −0.14 −0.01 D95 K −0.14 −0.14 −0.01 D95 L −0.14 −0.14 −0.01 D95 N −0.14 −0.14 −0.01 D95 Q −0.14 −0.14 −0.01 D95 R −0.14 −0.14 −0.01 D95 S −0.14 −0.14 −0.01 D95 T −0.14 −0.14 −0.01 D95 V −0.14 −0.14 −0.01 D95 W −0.14 −0.14 −0.01 D95 Y −0.14 −0.14 −0.01 T96 A 0.36 4.20 1.32 T96 C 0.44 3.76 0.79 T96 F 0.53 1.24 0.69 T96 G 0.78 1.28 1.03 T96 I 0.95 −0.22 0.88 T96 L 0.92 1.93 0.93 T96 M 0.39 2.53 0.80 T96 P −0.11 0.89 0.35 T96 R 0.17 0.14 0.50 T96 S 1.04 0.79 1.05 T96 T 1.00 1.00 1.00 T96 V 0.81 0.59 1.12 T96 W 0.38 −4.29 0.51 T96 Y 0.38 −3.73 0.59 K97 A 0.01 0.23 1.11 K97 D −0.23 −0.17 −0.01 K97 G 0.84 −0.64 0.39 K97 I 0.74 −0.55 0.47 K97 K 1.00 1.00 1.00 K97 L 0.38 −0.28 0.30 K97 M 0.02 0.22 0.95 K97 P 0.16 0.27 0.36 K97 Q 1.14 0.00 0.73 K97 R 2.80 0.59 1.02 K97 S 0.28 −0.46 0.58 K97 T 0.22 −0.42 0.51 K97 V 0.31 −0.45 0.51 K97 W 0.42 −2.32 0.13 K97 Y 0.29 −0.65 0.38 A98 A 1.00 1.00 1.00 A98 C 1.30 1.42 1.00 A98 D 1.11 2.19 0.81 A98 G 1.57 0.56 0.97 A98 H 2.09 0.92 0.82 A98 I 2.05 0.65 0.72 A98 L 2.22 1.47 0.71 A98 N 1.24 1.40 1.01 A98 P 1.10 1.26 0.90 A98 S 1.73 0.65 1.17 A98 T 1.72 0.27 1.03 A98 Y 2.02 1.15 0.87 Y99 A 0.66 0.82 1.29 Y99 G 0.83 0.70 1.23 Y99 H 0.77 0.59 1.30 Y99 I 0.81 0.61 1.11 Y99 L 0.66 0.86 1.39 Y99 P 0.89 0.81 1.00 Y99 R 0.61 0.29 0.97 Y99 S 0.72 0.37 1.45 Y99 V 0.61 0.31 1.28 Y99 W 0.68 0.57 1.20 Y99 Y 1.00 1.00 1.00 F100 A 0.78 2.02 0.93 F100 C 0.73 1.28 0.78 F100 D 0.38 −0.03 0.33 F100 E 1.01 0.15 0.83 F100 F 1.00 1.00 1.00 F100 K 0.65 −0.60 0.53 F100 M 0.79 2.19 1.20 F100 N 0.91 1.45 1.12 F100 S 0.87 0.85 1.02 F100 T 0.95 1.42 0.71 F100 W 1.08 −0.03 1.06 R101 C 0.71 0.95 0.96 R101 D 0.85 0.80 1.02 R101 F 0.84 0.97 0.66 R101 I 0.79 0.96 0.68 R101 K 1.24 0.07 0.90 R101 L 0.83 1.12 1.33 R101 N 0.72 0.92 1.11 R101 P 0.50 0.86 0.75 R101 Q 0.86 0.11 1.03 R101 R 1.00 1.00 1.00 R101 V 0.74 0.44 0.90 R101 W 0.95 0.00 0.89 R101 Y 0.74 0.80 0.67 R102 A 0.19 1.79 0.98 R102 C 0.22 0.36 0.78 R102 D 0.01 0.68 0.26 R102 F 0.46 0.23 0.31 R102 G 0.44 0.27 0.43 R102 L 0.33 1.64 0.95 R102 P −0.07 0.89 0.26 R102 Q 0.67 1.19 1.09 R102 R 1.00 1.00 1.00 R102 S 0.46 0.96 0.98 R102 V 0.28 0.61 0.80 R102 W 0.29 −1.03 0.34 R102 Y 0.40 1.29 0.70 T103 A 0.97 −9.64 0.89 T103 C 0.90 −6.91 0.89 T103 F 0.74 −3.39 0.85 T103 G 1.11 −5.27 1.20 T103 H 0.99 −4.15 1.14 T103 I 1.08 −5.15 0.89 T103 K 1.09 −4.36 1.05 T103 L 1.05 −1.86 0.88 T103 N 0.77 −6.03 1.07 T103 P 0.69 −5.11 1.01 T103 R 0.87 −6.30 0.96 T103 S 0.92 −1.36 1.14 T103 T 1.00 1.00 1.00 T103 V 0.95 −1.95 0.90 T103 W 1.26 −2.60 0.77 T103 Y 1.19 −4.68 0.88 P104 A −0.41 −0.19 −0.04 P104 C 1.95 1.83 1.34 P104 E 1.84 1.97 1.37 P104 F 1.79 0.86 0.67 P104 G 2.67 0.98 1.25 P104 H 2.84 1.03 1.11 P104 I 2.43 2.05 1.07 P104 L −0.41 −0.19 −0.04 P104 M 1.09 2.24 1.01 P104 N 1.62 1.44 1.32 P104 P 1.00 1.00 1.00 P104 Q 1.34 0.85 1.24 P104 R 1.62 −0.39 0.83 P104 S 2.48 0.53 1.44 P104 T 2.70 0.33 1.29 P104 V 2.59 1.02 1.40 P104 W 2.05 0.23 0.59 L105 A −0.11 −0.18 −0.02 L105 C 1.56 1.92 1.05 L105 E −0.11 0.53 0.26 L105 F 1.30 1.73 0.95 L105 G 1.08 1.40 1.07 L105 H 0.85 1.23 1.07 L105 L 1.00 1.00 1.00 L105 M −0.11 −0.18 −0.02 L105 P 1.71 0.90 1.00 L105 Q 0.94 1.04 1.03 L105 R 0.99 1.25 0.94 L105 S 0.93 0.61 0.95 L105 T 0.92 0.64 1.00 L105 V 0.15 −0.97 0.37 L105 W 1.28 1.71 0.78 L105 Y 0.72 0.62 1.18 D106 A 0.72 1.13 0.69 D106 C 1.01 1.10 0.80 D106 D 1.00 1.00 1.00 D106 E 1.08 1.09 1.02 D106 F 1.02 1.45 0.34 D106 G 1.18 1.45 0.67 D106 H 1.09 1.18 0.66 D106 I 1.04 0.92 0.45 D106 K 1.28 1.24 0.68 D106 L 1.20 1.00 0.56 D106 M 0.73 0.86 0.77 D106 N 0.92 0.64 0.91 D106 P −0.17 0.63 0.18 D106 Q 0.92 0.62 0.94 D106 R 0.98 0.56 0.91 D106 S 0.98 1.02 0.81 D106 T 1.06 1.38 0.64 D106 V 0.98 1.68 0.61 D106 W 0.78 1.07 0.34 I107 A 0.81 0.80 0.83 I107 C 0.95 1.41 1.00 I107 E 2.55 −0.28 0.21 I107 F 0.99 −0.02 0.19 I107 G 1.76 −10.12 0.25 I107 H −0.07 −0.20 −0.02 I107 I 1.00 1.00 1.00 I107 L 0.96 1.04 0.52 I107 N 1.81 0.93 0.56 I107 P 0.65 0.32 0.40 I107 Q 0.53 −0.02 0.43 I107 R 0.08 −2.75 0.28 I107 S 2.04 1.33 1.05 I107 T 0.64 1.53 0.95 I107 V 1.00 0.97 1.04 I107 W −0.07 −0.20 −0.02 I107 Y 0.49 0.52 0.23 A108 A −0.12 −0.07 −0.02 A108 D −0.12 −0.07 −0.02 A108 E 0.14 0.61 0.25 A108 F −0.12 −0.07 −0.02 A108 G 0.99 1.13 1.15 A108 H −0.12 −0.07 −0.02 A108 I −0.12 −0.07 −0.02 A108 K 0.60 2.97 0.31 A108 L 1.41 2.56 0.20 A108 N −0.12 −0.07 −0.02 A108 P −0.12 −0.07 −0.02 A108 Q 0.58 0.73 0.98 A108 R −0.12 −0.07 −0.02 A108 S 0.94 1.00 1.14 A108 T 1.05 0.87 1.08 A108 V 0.76 0.95 0.99 L109 A 0.34 0.32 1.07 L109 D 1.00 0.11 1.15 L109 E 0.74 0.19 1.24 L109 F 0.83 0.32 1.11 L109 G 0.82 0.51 0.88 L109 H 0.85 0.22 1.06 L109 I 1.05 0.14 1.21 L109 L 1.00 1.00 1.00 L109 M 0.74 0.63 1.00 L109 N 1.52 0.66 1.13 L109 P 0.79 0.43 0.35 L109 Q 1.18 0.22 1.08 L109 R 0.48 0.21 0.95 L109 S 0.79 0.38 0.94 L109 T 0.63 0.79 0.87 L109 V 0.52 0.54 1.06 L109 W 1.30 −0.02 0.88 L109 Y 1.16 0.83 0.79 G110 A 0.91 1.01 0.88 G110 C 0.35 1.43 0.56 G110 D 0.76 1.40 0.87 G110 E 0.26 1.76 0.46 G110 F 0.04 2.29 0.30 G110 G 1.00 1.00 1.00 G110 H 0.63 0.73 0.46 G110 I 0.06 0.23 0.32 G110 L −0.20 −0.12 −0.02 G110 M 0.16 0.82 0.34 G110 N 0.70 0.77 0.89 G110 P 0.02 0.22 0.50 G110 Q 0.44 0.34 0.77 G110 R 0.05 0.48 0.45 G110 S 0.79 0.30 1.01 G110 T 0.45 −0.05 0.42 G110 W −0.20 −1.18 0.20 G110 Y 0.01 −0.88 0.40 M111 A 0.65 1.02 0.89 M111 C 0.92 1.01 0.95 M111 D −0.27 0.79 0.37 M111 E 0.25 0.67 0.56 M111 F 1.47 0.78 0.75 M111 G 0.85 0.32 0.44 M111 H 0.98 0.19 0.40 M111 I 1.95 1.03 0.91 M111 K 1.98 0.71 0.58 M111 L 1.55 0.67 0.93 M111 M 1.00 1.00 1.00 M111 N 0.49 1.31 0.79 M111 P −0.27 0.57 0.39 M111 R 0.27 −0.99 0.34 M111 S 1.03 0.14 0.52 M111 T 1.49 0.76 0.77 M111 V 1.47 0.93 0.88 M111 W 0.96 1.23 0.30 M111 Y 1.43 1.06 0.65 S112 A 0.58 0.94 0.98 S112 E 0.71 1.16 1.05 S112 F 0.37 0.88 0.61 S112 H 1.00 0.38 0.93 S112 K 0.84 0.68 0.92 S112 L 1.03 1.00 0.80 S112 M 0.43 0.56 0.98 S112 N 0.52 0.85 1.09 S112 P −0.19 −0.82 0.33 S112 R 0.20 −0.44 0.99 S112 S 1.00 1.00 1.00 S112 T 0.95 0.72 0.87 S112 V 0.86 0.48 0.73 S112 W 0.74 0.58 0.85 S112 Y 0.68 −0.10 0.90 V113 A 0.71 1.31 0.70 V113 C 0.87 0.94 1.06 V113 D 0.78 0.87 0.97 V113 E 0.91 0.94 0.99 V113 F 1.05 0.96 0.80 V113 G 0.96 0.58 0.89 V113 H 1.34 0.76 0.84 V113 K 1.19 0.72 0.92 V113 L 1.50 0.85 0.85 V113 M 0.78 1.06 0.93 V113 N 0.88 1.22 1.01 V113 P 0.72 1.14 0.65 V113 Q 1.03 1.11 0.94 V113 R 1.13 1.11 0.82 V113 S 0.80 0.57 0.91 V113 T 0.94 0.86 0.89 V113 V 1.00 1.00 1.00 V113 W 0.91 0.80 0.76 V113 Y 1.11 0.98 0.85 L114 A 0.78 1.07 1.03 L114 C 0.78 1.14 1.10 L114 E 0.32 −0.14 0.42 L114 F −0.11 −0.21 −0.02 L114 G 0.96 1.14 0.78 L114 H 0.92 −0.55 0.21 L114 I 0.97 1.17 0.86 L114 K −0.11 −0.21 −0.02 L114 L 1.00 1.00 1.00 L114 M 0.73 1.28 1.00 L114 N 0.65 0.77 0.95 L114 P 0.30 0.28 0.42 L114 Q 0.59 0.12 0.68 L114 R −0.11 −0.21 −0.02 L114 S 0.87 0.55 0.72 L114 T 0.88 1.05 0.82 L114 V 0.91 0.60 0.84 L114 W −0.11 −0.21 −0.02 L114 Y −0.11 −0.21 −0.02 V115 A 0.60 1.19 1.11 V115 C 0.73 1.08 1.14 V115 D −0.15 2.21 0.19 V115 F 0.54 1.69 0.32 V115 G 1.09 1.76 0.43 V115 H −0.15 −0.13 −0.02 V115 I 1.05 0.99 1.14 V115 K −0.15 −0.13 −0.02 V115 L 1.12 1.30 1.02 V115 M 0.48 1.32 1.05 V115 P −0.15 2.21 0.26 V115 Q −0.15 1.15 0.32 V115 R 0.10 1.63 0.21 V115 S 0.95 1.14 0.72 V115 T 1.15 1.28 0.72 V115 V 1.00 1.00 1.00 V115 W 1.23 2.48 0.17 V115 Y 1.03 2.07 0.28 T116 A 1.01 0.95 1.08 T116 C 0.89 1.05 1.30 T116 E 0.86 0.91 1.29 T116 G 1.10 0.90 1.44 T116 H 1.00 1.08 1.48 T116 I 0.80 0.76 0.82 T116 L 0.77 0.68 1.03 T116 M 0.83 1.39 1.28 T116 N 0.93 1.05 1.68 T116 P 0.74 0.84 0.99 T116 Q 0.95 0.77 1.29 T116 R 0.64 0.62 1.03 T116 S 0.88 0.96 1.24 T116 T 1.00 1.00 1.00 T116 V 0.86 0.57 0.85 T116 W 0.89 0.75 0.96 T116 Y 0.90 0.47 1.09 Q117 A 2.05 1.73 1.03 Q117 E 1.15 1.21 1.10 Q117 F 1.57 1.02 0.61 Q117 G 2.08 0.79 0.97 Q117 H 2.33 1.12 1.12 Q117 M 1.54 1.89 0.87 Q117 P −0.25 1.13 0.61 Q117 Q 1.00 1.00 1.00 Q117 R 1.56 1.05 1.00 Q117 S 1.95 0.87 1.13 Q117 T 2.23 1.10 1.06 Q117 V 2.15 0.76 0.67 Q117 W 2.16 0.71 0.57 Q117 Y 2.23 1.13 0.76 V118 A 0.84 0.85 1.20 V118 C 0.78 1.14 1.28 V118 D −0.14 0.40 0.38 V118 E −0.14 −0.43 0.37 V118 F 0.86 1.00 0.89 V118 G 1.08 0.56 0.67 V118 I 0.96 0.55 1.01 V118 K 1.13 −2.50 0.28 V118 L 0.93 1.05 0.93 V118 M 0.60 0.93 0.90 V118 P 0.12 0.22 0.52 V118 Q 0.38 1.50 0.57 V118 R 0.36 0.07 0.46 V118 S 0.95 0.82 0.96 V118 T 0.99 0.92 0.90 V118 V 1.00 1.00 1.00 V118 W 0.83 −1.28 0.42 V118 Y 1.25 1.34 0.60 L119 A 0.81 1.02 1.18 L119 C 0.76 0.24 1.18 L119 D 0.24 0.28 0.97 L119 E 0.45 0.32 1.04 L119 F 0.56 −0.61 0.93 L119 G 0.93 −0.06 0.97 L119 H 0.91 0.46 0.89 L119 I 0.90 0.43 1.06 L119 L 1.00 1.00 1.00 L119 N 0.58 0.11 1.14 L119 P −0.14 −0.01 0.71 L119 R 0.43 −0.66 1.00 L119 S 0.83 −0.17 1.05 L119 T 0.97 0.10 0.94 L119 V 0.89 0.15 1.04 L119 W 0.77 0.20 0.88 L119 Y 0.77 0.56 0.89 T120 A 0.25 0.66 1.09 T120 C 0.75 0.92 1.14 T120 E 0.58 1.53 1.19 T120 H 0.88 0.50 1.07 T120 I 0.91 1.56 1.00 T120 K 0.87 1.09 1.12 T120 L 0.80 1.26 1.00 T120 M 0.05 1.22 0.98 T120 N 0.37 1.42 1.10 T120 P 0.07 −0.45 0.82 T120 Q 0.26 0.78 1.05 T120 R 0.24 0.60 0.99 T120 S 1.09 1.07 1.35 T120 T 1.00 1.00 1.00 T120 V 0.26 1.07 0.93 T120 Y 0.57 1.61 1.01 S121 A 1.12 1.55 1.10 S121 C 1.18 1.64 1.09 S121 E 0.89 1.04 1.01 S121 G 1.20 0.99 1.07 S121 K 1.24 0.78 1.04 S121 L 1.35 1.49 1.12 S121 N 1.14 1.06 1.17 S121 P 0.83 0.38 0.92 S121 Q 0.92 1.09 1.01 S121 R 1.26 0.70 1.06 S121 S 1.00 1.00 1.00 S121 T 1.13 1.26 0.93 S121 V 1.12 1.59 0.97 S121 W 1.33 0.77 0.91 A122 A 1.00 1.00 1.00 A122 D 0.26 0.06 0.77 A122 E 0.71 0.47 1.04 A122 F 0.97 0.15 0.87 A122 G 0.93 −0.42 0.85 A122 H 1.14 0.17 1.00 A122 I 1.13 0.65 1.04 A122 K 1.08 0.45 0.96 A122 L 0.93 1.02 1.07 A122 M 0.81 0.94 1.06 A122 N 0.83 0.70 1.11 A122 P 0.61 0.55 1.07 A122 Q 0.69 0.74 1.02 A122 R 0.71 0.40 0.94 A122 S 1.03 0.43 1.05 A122 T 1.08 0.52 0.97 A122 V 1.04 0.89 1.05 A122 W 0.99 0.86 0.88 G123 A 0.89 1.19 0.96 G123 C 0.95 0.30 0.92 G123 D 1.73 0.84 0.90 G123 E 1.13 0.56 0.96 G123 F 0.84 0.80 0.85 G123 G 1.00 1.00 1.00 G123 H 1.00 0.74 0.84 G123 K 0.97 1.12 0.93 G123 L 0.99 1.38 0.79 G123 M 0.84 1.38 0.85 G123 N 0.89 0.71 0.92 G123 P 1.32 0.81 0.89 G123 Q 0.01 0.31 0.37 G123 R 0.66 0.60 0.83 G123 T 1.06 0.54 0.85 G123 V 1.40 0.59 0.89 G123 W 0.95 1.39 0.77 G123 Y 0.96 1.24 0.87 G124 A 0.84 0.03 1.20 G124 C 0.72 0.67 1.07 G124 D 0.76 0.64 0.99 G124 F 1.32 0.95 0.70 G124 G 1.00 1.00 1.00 G124 H 1.59 −0.10 0.98 G124 I 1.85 −0.08 0.92 G124 L 1.92 0.54 0.98 G124 M 0.97 −0.05 1.36 G124 N 0.98 0.60 1.18 G124 P −0.11 −0.08 0.37 G124 Q 1.12 0.21 1.02 G124 R 1.14 0.41 0.88 G124 S 1.27 0.56 1.00 G124 T 1.64 0.32 0.97 G124 V 1.44 0.33 0.93 G124 W 0.73 −0.31 0.84 G124 Y 1.23 0.56 0.66 V125 A 1.69 0.93 0.91 V125 C 0.96 0.54 0.67 V125 D 1.24 0.54 0.76 V125 E 0.81 0.39 0.73 V125 F 0.96 0.63 0.77 V125 G 2.95 1.09 0.60 V125 I 1.01 0.94 1.05 V125 P 1.50 0.62 0.83 V125 R 1.30 0.47 0.82 V125 S 1.94 0.79 0.75 V125 V 1.00 1.00 1.00 V125 W 0.37 0.25 0.48 V125 Y 1.08 0.81 0.82 G126 A 0.96 0.55 1.02 G126 C 0.35 0.98 0.96 G126 D 0.33 1.22 0.93 G126 E 0.67 0.60 1.02 G126 G 1.00 1.00 1.00 G126 I 0.84 0.01 0.81 G126 L 1.17 0.54 0.90 G126 M 0.43 1.17 0.92 G126 N 0.38 0.85 1.04 G126 P 1.17 0.67 0.82 G126 R 0.43 0.76 0.89 G126 S 0.76 0.90 0.90 G126 T 1.58 0.74 0.90 G126 V 0.89 0.18 0.84 G126 Y 0.54 0.23 0.82 T127 A 0.73 1.10 1.10 T127 C 0.76 0.65 1.04 T127 D 0.46 0.62 1.03 T127 E 0.40 −0.01 1.03 T127 G 0.95 0.71 1.04 T127 H 1.57 0.60 0.99 T127 I 1.06 0.20 0.91 T127 L 0.90 −0.03 0.94 T127 M 0.79 0.64 1.02 T127 P 0.14 0.77 0.95 T127 Q 0.55 0.15 0.86 T127 S 1.05 0.83 1.08 T127 T 1.00 1.00 1.00 T127 V 1.07 0.68 1.06 T128 A 0.76 1.31 1.23 T128 D 0.78 0.66 1.14 T128 F 0.79 1.71 1.01 T128 H 0.99 1.08 1.19 T128 K 1.06 1.57 1.10 T128 L 1.06 1.72 0.97 T128 M 0.72 1.06 1.28 T128 N 0.70 1.36 1.29 T128 P 0.87 1.16 1.18 T128 Q 0.78 1.34 1.24 T128 R 0.87 1.70 1.03 T128 S 0.92 1.27 1.07 T128 T 1.00 1.00 1.00 T128 V 0.98 1.15 1.05 T128 W 0.92 1.23 0.95 T128 Y 0.95 1.81 0.96 Y129 A 0.64 0.17 1.39 Y129 C 0.66 0.61 1.42 Y129 D 0.35 0.23 1.35 Y129 F 0.71 0.71 1.44 Y129 G 0.39 −0.56 1.10 Y129 K 0.31 −0.29 1.00 Y129 L 0.78 0.27 1.22 Y129 M 0.68 0.21 1.28 Y129 N 0.46 0.53 1.24 Y129 P 0.15 0.59 1.11 Y129 R 0.38 0.18 1.00 Y129 S 0.67 0.69 1.08 Y129 T 0.46 0.14 1.00 Y129 V 0.24 −0.29 1.00 Y129 W 0.47 −0.15 1.01 Y129 Y 1.00 1.00 1.00 P130 A 0.82 0.44 1.03 P130 C 0.95 0.64 0.93 P130 E 1.00 0.22 1.08 P130 F 1.08 0.48 0.89 P130 G 1.16 −0.19 1.11 P130 H 1.17 0.01 1.00 P130 I 1.12 0.41 0.94 P130 K 1.16 0.55 1.05 P130 L 1.12 0.09 0.98 P130 M 0.66 0.76 1.03 P130 P 1.00 1.00 1.00 P130 R 1.11 0.53 0.95 P130 S 1.16 −0.14 0.96 P130 T 1.19 −0.06 0.96 P130 V 1.15 0.37 0.94 P130 W 1.15 0.28 0.80 A131 A 1.00 1.00 1.00 A131 D 1.31 0.40 0.80 A131 E 1.36 0.97 0.88 A131 G 1.66 0.87 0.83 A131 H 1.72 0.82 0.75 A131 L 1.83 0.59 0.73 A131 P 1.52 0.71 0.94 A131 Q 1.29 0.74 0.69 A131 R 1.76 1.04 0.61 A131 S 1.48 0.68 0.87 A131 V 1.59 0.78 0.89 A131 W 1.61 −0.42 0.65 A131 Y 1.50 0.48 0.73 P132 A 0.49 6.08 0.94 P132 C 0.49 5.68 0.94 P132 D −0.11 −7.16 0.62 P132 E 0.19 3.02 0.80 P132 F 0.76 −1.33 0.49 P132 G 0.83 4.98 0.79 P132 H 0.50 −1.95 0.68 P132 I 0.58 −3.19 0.64 P132 L 0.87 2.24 0.67 P132 N 0.30 1.05 0.83 P132 P 0.09 6.91 1.03 P132 Q 0.41 6.15 0.91 P132 R 0.02 −2.19 0.65 P132 S 1.13 5.05 0.96 P132 T 0.85 −2.01 0.75 P132 V 0.85 −2.29 0.78 P132 W 0.77 −2.64 0.37 P132 Y 1.57 4.78 0.60 K133 A 0.67 0.10 1.01 K133 C 0.56 −0.11 0.72 K133 E 0.63 0.76 1.01 K133 F 0.86 0.59 0.73 K133 G 0.97 0.31 0.87 K133 H 1.02 0.31 0.87 K133 I 0.89 0.45 0.78 K133 K 1.00 1.00 1.00 K133 L 1.05 1.92 0.76 K133 M 0.68 0.33 0.98 K133 P 0.39 0.71 0.89 K133 Q 0.69 0.52 1.13 K133 R 0.78 0.83 1.01 K133 S 0.84 0.58 1.02 K133 T 0.93 0.39 0.97 K133 V 0.90 0.23 0.87 K133 W 0.97 0.99 0.46 K133 Y 1.12 1.44 0.75 V134 A 0.75 1.64 0.87 V134 C 0.77 1.37 0.91 V134 D −0.08 −0.08 −0.02 V134 G 1.71 1.42 0.45 V134 I 1.12 0.89 0.99 V134 K −0.08 −0.08 −0.02 V134 L 1.13 1.45 0.78 V134 M 0.82 1.89 0.83 V134 N 1.18 2.80 0.25 V134 P −0.08 1.71 0.43 V134 Q 0.04 0.79 0.44 V134 R −0.08 −0.08 −0.02 V134 S 1.16 1.44 0.62 V134 T 1.25 0.86 0.82 V134 V 1.00 1.00 1.00 V134 W −0.08 −0.08 −0.02 V134 Y −0.08 −0.08 −0.02 L135 D −0.13 2.90 0.27 L135 E −0.13 0.63 0.39 L135 F 0.34 −0.03 0.45 L135 G 0.33 −1.71 0.28 L135 K 0.66 −1.23 0.28 L135 L 1.00 1.00 1.00 L135 M 0.77 0.78 1.01 L135 P −0.13 −1.31 0.22 L135 Q 0.34 0.17 0.66 L135 R 0.06 −1.41 0.25 L135 S 0.50 −0.65 0.44 L135 T 0.73 −0.42 0.50 L135 V 0.83 0.43 0.82 L135 W 0.71 −0.42 0.36 V136 A 0.60 1.60 0.66 V136 C 0.57 1.23 0.87 V136 E −0.09 0.20 0.25 V136 L 0.98 1.13 1.03 V136 N −0.09 0.40 0.26 V136 P −0.09 −0.12 0.52 V136 R −0.09 −0.12 −0.02 V136 T 1.13 1.13 0.68 V136 V 1.00 1.00 1.00 V136 W −0.09 −0.12 −0.02 V137 A 1.07 1.46 0.64 V137 C 0.98 1.42 0.85 V137 D −0.17 −0.23 −0.01 V137 E −0.17 −0.23 −0.01 V137 F −0.17 −0.23 −0.01 V137 G 1.02 0.26 0.13 V137 I 0.98 0.70 0.83 V137 L 1.09 1.27 0.82 V137 M 1.22 1.13 0.89 V137 N 0.46 −1.29 0.15 V137 P −0.17 −0.23 −0.01 V137 R −0.17 −0.23 −0.01 V137 S 0.96 0.29 0.50 V137 T 1.08 0.93 0.73 V137 V 1.00 1.00 1.00 V137 W −0.17 −0.23 −0.01 V137 Y −0.17 −0.23 −0.01 S138 A 0.69 1.28 1.44 S138 C 0.64 1.18 1.17 S138 E −0.13 −0.19 −0.02 S138 F −0.13 −0.19 −0.02 S138 G 1.05 1.11 1.09 S138 H −0.13 −0.19 −0.02 S138 I 1.15 0.35 0.56 S138 L −0.13 −0.19 −0.02 S138 M −0.13 −0.19 −0.02 S138 N 0.62 1.31 0.77 S138 P 0.54 1.39 0.45 S138 Q −0.13 −0.19 −0.02 S138 R −0.13 −0.19 −0.02 S138 S 1.00 1.00 1.00 S138 V 1.00 0.69 0.67 S138 W −0.13 −0.19 −0.02 S138 Y −0.13 −0.19 −0.02 P139 C 0.08 −0.12 0.18 P139 D −0.13 −1.44 0.15 P139 E −0.13 −5.11 0.19 P139 F −0.13 −4.13 0.16 P139 G 0.50 −3.08 0.23 P139 H −0.13 −6.03 0.19 P139 I −0.13 −3.71 0.21 P139 K −0.13 −4.09 0.12 P139 L −0.13 −0.17 −0.02 P139 N −0.13 −2.11 0.16 P139 P 1.00 1.00 1.00 P139 Q −0.13 −0.32 0.18 P139 R 0.37 −1.04 0.23 P139 S 0.88 −0.52 0.43 P139 T 0.01 −3.48 0.15 P139 V −0.13 −1.70 0.17 P139 W −0.13 −0.17 −0.02 P139 Y −0.13 −0.17 −0.02 P140 A 1.90 1.83 0.61 P140 C 0.39 1.07 0.40 P140 D −0.45 −0.23 −0.02 P140 F −0.45 2.89 0.19 P140 G 0.96 3.11 0.20 P140 H 0.59 2.25 0.23 P140 I 0.45 −1.03 0.24 P140 K −0.45 −0.23 −0.02 P140 L −0.45 −0.23 −0.02 P140 M −0.45 −0.23 −0.02 P140 P 1.00 1.00 1.00 P140 Q −0.45 −1.32 0.32 P140 R −0.45 −2.74 0.25 P140 S 1.31 −1.22 0.43 P140 T 1.74 −0.78 0.29 P140 V 0.50 −1.12 0.34 P140 W 0.50 −0.97 0.17 P140 Y 0.32 −1.90 0.24 P141 A 1.10 1.08 1.13 P141 G 1.64 −0.05 1.02 P141 H 2.07 0.79 0.93 P141 I 2.29 0.38 0.90 P141 L 2.32 0.65 0.74 P141 N 1.32 0.97 0.96 P141 P 1.00 1.00 1.00 P141 Q 1.39 0.37 0.88 P141 R 1.65 −0.26 0.61 P141 S 1.70 0.02 0.90 P141 T 1.84 0.12 0.82 P141 V 1.96 0.16 0.72 L142 A 0.80 0.56 0.67 L142 C 0.74 0.70 0.78 L142 D −0.12 −0.13 −0.01 L142 F 1.05 0.54 0.46 L142 G −0.12 −0.13 −0.01 L142 I 0.64 0.28 1.05 L142 K 1.60 0.66 0.23 L142 L 1.00 1.00 1.00 L142 M −0.12 −0.13 −0.01 L142 N −0.12 −0.13 −0.01 L142 P 0.54 0.44 0.48 L142 Q 0.67 0.33 0.49 L142 R −0.12 −0.13 −0.01 L142 S 0.84 0.31 0.65 L142 T −0.12 −0.13 −0.01 L142 V 0.84 0.33 0.82 L142 W 2.41 −1.89 0.16 A143 A 1.00 1.00 1.00 A143 C 1.39 1.07 0.81 A143 D 1.45 1.22 0.71 A143 E 1.43 1.13 0.71 A143 F 1.56 0.68 0.99 A143 G 1.48 0.42 1.17 A143 H 2.90 1.36 0.70 A143 K 3.16 1.37 0.62 A143 L 2.51 1.28 0.71 A143 N 1.30 0.82 0.79 A143 P 1.53 0.39 0.63 A143 Q 1.74 0.81 0.72 A143 R 2.15 0.99 0.62 A143 S 1.77 0.63 0.98 A143 T 2.18 0.97 0.74 A143 V 2.45 0.99 0.81 A143 W 2.27 −0.21 0.37 P144 A 1.09 0.79 0.91 P144 D 1.45 1.38 0.60 P144 F 1.82 1.08 0.66 P144 G 1.45 0.62 0.78 P144 H 1.94 1.60 0.66 P144 K 2.09 1.09 0.67 P144 L 1.43 1.15 0.86 P144 M 1.24 1.01 0.76 P144 N 1.44 1.49 0.74 P144 P 1.00 1.00 1.00 P144 Q 1.37 1.08 0.77 P144 R 1.76 1.14 0.68 P144 S 1.69 0.92 0.77 P144 T 1.46 0.81 0.80 P144 Y 2.34 1.65 0.70 M145 A 0.44 0.79 0.94 M145 C 1.02 0.93 0.94 M145 E 0.28 0.48 0.74 M145 F 1.49 0.77 0.95 M145 G 0.48 0.26 0.92 M145 I 0.79 0.53 1.16 M145 L 1.72 0.61 1.07 M145 M 1.00 1.00 1.00 M145 P 0.64 0.78 0.78 M145 Q 0.68 0.57 0.86 M145 R 1.15 0.69 0.78 M145 S 0.64 0.78 0.91 M145 T 1.01 0.79 0.91 M145 V 0.72 0.63 1.00 M145 W 1.15 −0.13 0.49 M145 Y 0.94 0.82 0.68 P146 A 0.20 1.36 0.73 P146 C 0.31 1.69 0.62 P146 F 0.55 1.53 0.51 P146 G 0.24 1.04 0.51 P146 H 0.50 1.57 0.56 P146 L 0.56 2.00 0.53 P146 M 0.39 1.23 0.79 P146 N 0.37 1.00 0.78 P146 P 1.00 1.00 1.00 P146 R 0.36 1.06 0.66 P146 S 0.46 0.96 0.82 P146 T 0.38 0.76 0.80 P146 V 0.55 0.77 0.89 P146 W 0.56 0.68 0.64 P146 Y 0.35 1.44 0.54 H147 A 1.28 0.98 0.96 H147 C 0.94 1.17 1.04 H147 D 0.95 1.18 1.00 H147 E 1.11 1.10 0.96 H147 G −0.12 −0.15 −0.02 H147 H 1.00 1.00 1.00 H147 I 0.89 0.92 0.89 H147 K 0.94 1.06 0.89 H147 L 0.69 1.29 1.09 H147 M 0.73 1.44 0.86 H147 N 0.84 1.25 0.98 H147 P 1.12 1.21 0.71 H147 Q 0.71 1.03 0.86 H147 R 0.89 0.94 0.69 H147 S 1.26 0.75 0.92 H147 T 1.20 0.84 0.85 H147 V 0.96 0.92 0.90 H147 W 0.88 1.05 0.79 H147 Y 0.75 1.12 0.94 P148 A 1.64 1.06 0.96 P148 D 1.03 1.34 0.74 P148 E 1.42 1.19 0.76 P148 F 1.37 1.50 0.64 P148 G 0.87 1.20 0.70 P148 K 1.79 1.30 0.72 P148 L 1.64 1.39 0.74 P148 P 1.00 1.00 1.00 P148 Q 1.33 0.98 0.81 P148 R 1.51 1.25 0.79 P148 S 1.46 1.21 0.74 P148 T 1.50 1.09 0.79 P148 V 2.43 1.04 0.76 P148 Y 1.46 1.37 0.72 W149 A 0.21 0.31 1.35 W149 C 0.18 0.12 0.93 W149 E 0.00 −0.04 0.85 W149 F 0.53 0.50 1.27 W149 G 0.26 0.45 1.39 W149 H 0.60 1.01 0.81 W149 I 0.21 0.24 0.83 W149 L 0.30 0.64 1.06 W149 M 0.33 0.49 1.32 W149 P −0.32 −0.16 0.92 W149 Q 0.11 0.40 1.10 W149 R 0.04 −0.32 0.67 W149 S 0.16 0.33 1.28 W149 T 0.26 0.44 0.84 W149 W 1.00 1.00 1.00 W149 Y 0.58 0.75 1.15 F150 A 0.01 0.54 1.70 F150 C 0.43 0.78 1.41 F150 E 1.23 0.73 1.32 F150 F 1.00 1.00 1.00 F150 G 0.14 0.46 1.13 F150 H 0.53 1.18 1.09 F150 I 0.40 0.78 1.19 F150 K 0.41 0.85 1.33 F150 L 1.29 1.30 1.14 F150 M 0.80 0.63 1.69 F150 N 0.55 0.36 1.52 F150 P 0.18 0.32 1.38 F150 T 0.37 0.58 1.27 F150 V 0.22 0.51 1.26 F150 W 0.19 0.62 1.26 F150 Y 0.72 1.07 1.24 Q151 A 1.29 2.93 0.46 Q151 C 1.05 2.55 0.38 Q151 D 1.47 2.81 0.83 Q151 E 1.14 2.07 0.99 Q151 F 0.31 −8.08 0.21 Q151 H 1.06 2.19 0.94 Q151 I 0.08 −2.76 0.16 Q151 K 1.07 2.19 1.04 Q151 L 0.40 −1.53 0.17 Q151 M 1.24 6.36 0.24 Q151 P 1.35 1.91 0.50 Q151 Q 1.00 1.00 1.00 Q151 R 1.36 2.32 0.68 Q151 S 1.05 2.25 0.86 Q151 T 1.24 2.37 0.64 Q151 V 0.36 −1.65 0.25 Q151 W 0.77 0.32 0.33 Q151 Y 1.01 2.75 0.41 L152 A 0.88 1.29 0.85 L152 C 1.00 1.14 0.87 L152 D 1.07 0.86 0.81 L152 E 1.08 1.23 0.93 L152 G 1.08 0.77 0.85 L152 H 1.09 0.92 0.93 L152 I 1.04 0.61 0.77 L152 K 1.21 0.91 0.93 L152 L 1.00 1.00 1.00 L152 M 0.99 1.10 0.82 L152 P 0.81 0.61 0.54 L152 Q 1.07 0.76 0.84 L152 R 1.20 0.91 0.89 L152 S 1.12 0.84 0.84 L152 T 1.12 0.69 0.82 L152 V 1.22 0.88 0.83 L152 W 1.18 1.55 0.74 L152 Y 1.09 1.37 0.89 I153 A 1.19 1.49 0.76 I153 F 1.23 1.75 0.47 I153 H 1.46 2.00 0.56 I153 I 1.00 1.00 1.00 I153 K 1.62 2.44 0.43 I153 L 1.27 1.50 0.82 I153 N 0.72 0.89 1.04 I153 P 0.25 1.87 0.31 I153 S 0.87 1.66 0.61 I153 T 1.27 1.62 0.64 I153 V 0.96 1.15 0.78 F154 D −0.19 −1.06 −0.02 F154 E −0.19 −1.06 −0.02 F154 F 1.00 1.00 1.00 F154 G −0.19 −0.64 0.17 F154 L −0.19 −1.06 −0.02 F154 P −0.19 −1.06 −0.02 F154 Q 0.39 0.97 0.45 F154 S 0.13 0.29 0.35 F154 T 0.12 −1.76 0.19 F154 V −0.19 −14.19 0.18 F154 Y 1.32 4.96 0.92 E155 A 0.99 2.59 0.83 E155 D 1.08 1.24 0.89 E155 E 1.00 1.00 1.00 E155 F 1.07 0.23 0.60 E155 G 1.17 1.12 0.82 E155 I 0.95 0.65 0.61 E155 K 1.23 1.33 0.83 E155 L 1.31 2.07 0.60 E155 M 0.73 2.91 0.74 E155 N 0.79 1.79 0.86 E155 P 0.79 2.60 0.65 E155 Q 0.90 0.69 0.87 E155 R 1.47 −0.07 0.71 E155 S 1.08 1.12 0.82 E155 T 1.49 1.19 0.76 E155 V 0.79 0.47 0.63 E155 Y 1.27 2.65 0.55 G156 A 0.99 1.21 0.88 G156 C 1.07 1.37 0.84 G156 D 0.96 1.62 0.93 G156 E 0.94 1.14 0.91 G156 F 0.90 0.73 0.78 G156 G 1.00 1.00 1.00 G156 H 1.04 1.40 0.84 G156 I 0.70 −0.08 0.44 G156 K 1.10 1.11 0.88 G156 L 0.90 0.94 0.74 G156 M 1.09 1.62 0.80 G156 N 1.07 1.38 0.97 G156 P 1.44 1.29 0.59 G156 R 1.05 1.21 0.80 G156 S 1.02 1.04 0.88 G156 T 1.15 1.53 0.79 G156 V 0.88 0.97 0.58 G156 W 0.89 0.90 0.56 G156 Y 0.96 1.40 0.80 G157 A 0.77 0.87 1.00 G157 C 0.96 0.61 0.92 G157 D 0.93 0.94 0.41 G157 E 0.98 0.84 0.61 G157 F 1.27 1.42 0.61 G157 G 1.00 1.00 1.00 G157 H 1.14 1.57 0.70 G157 I 1.11 1.33 0.36 G157 K 1.28 1.47 0.46 G157 M 0.96 0.85 0.70 G157 P 0.86 0.01 0.31 G157 R 1.51 −0.10 0.42 G157 S 1.30 0.19 0.93 G157 T 1.74 0.99 0.68 G157 V 1.23 0.40 0.59 E158 A 1.45 1.28 0.91 E158 C 1.46 1.37 0.67 E158 D 1.35 0.89 0.82 E158 E 1.00 1.00 1.00 E158 F 2.06 1.77 0.46 E158 H 2.40 1.01 0.59 E158 I 1.38 0.94 0.76 E158 K 2.08 1.88 0.62 E158 L 1.59 1.96 0.70 E158 M 1.39 1.73 0.71 E158 N 1.41 1.58 0.82 E158 P 1.41 1.19 0.85 E158 Q 1.49 1.24 0.85 E158 R 1.99 1.29 0.62 E158 S 1.57 1.27 0.82 E158 T 1.45 0.91 0.77 E158 V 1.52 0.89 0.81 E158 W 1.77 1.31 0.67 E158 Y 1.77 2.48 0.57 Q159 A 1.08 0.28 1.13 Q159 C 1.13 0.31 0.79 Q159 D 1.09 0.63 0.90 Q159 E 0.99 0.97 1.14 Q159 G 0.96 0.72 1.03 Q159 H 0.96 1.48 0.90 Q159 L 1.02 0.70 0.83 Q159 M 1.07 0.84 0.83 Q159 P 1.06 0.49 0.81 Q159 Q 1.00 1.00 1.00 Q159 R 1.15 0.74 0.76 Q159 S 1.10 0.73 0.81 K160 A 0.39 1.14 0.86 K160 C 0.48 1.29 0.77 K160 D −0.15 1.19 0.40 K160 G 0.91 0.30 0.56 K160 H 0.98 0.57 0.65 K160 I 0.97 1.00 0.78 K160 K 1.00 1.00 1.00 K160 L 0.97 0.95 0.77 K160 M 0.31 1.47 0.78 K160 N 0.37 1.12 0.65 K160 P −0.15 1.66 0.31 K160 Q 0.45 1.41 0.75 K160 R 0.83 1.15 0.76 K160 S 0.85 0.70 0.74 K160 W 0.89 −0.34 0.21 T161 C 0.84 0.56 1.01 T161 D −0.14 −0.21 −0.02 T161 E −0.14 −0.21 −0.02 T161 G 0.92 0.43 0.94 T161 H 1.82 −0.15 0.42 T161 I 1.40 0.98 0.91 T161 L 1.25 1.16 0.81 T161 M 0.57 1.72 0.83 T161 N 0.80 −0.86 0.32 T161 P −0.14 −0.21 −0.02 T161 Q 1.04 1.50 0.90 T161 R 3.61 −1.68 0.42 T161 S 0.92 0.57 0.98 T161 T 1.00 1.00 1.00 T161 V 1.27 1.24 1.00 T161 W 1.41 0.00 0.52 T161 Y 2.40 2.62 0.23 T162 C 0.95 3.57 1.17 T162 F 0.99 3.23 1.05 T162 G 1.00 1.82 0.88 T162 H 1.02 3.91 1.08 T162 I 0.99 2.21 1.16 T162 K 1.22 3.13 0.98 T162 L 1.00 3.59 1.05 T162 M 0.77 3.49 0.89 T162 N 0.83 3.84 0.98 T162 P 0.96 4.37 0.81 T162 Q 0.93 2.45 0.89 T162 R 1.17 1.23 0.80 T162 S 0.98 2.01 0.97 T162 T 1.00 1.00 1.00 T162 W 1.15 2.04 0.85 T162 Y 1.03 2.89 1.03 E163 A 1.11 1.79 0.73 E163 C 1.11 1.08 0.67 E163 D 0.90 1.08 0.82 E163 E 1.00 1.00 1.00 E163 F 1.07 0.27 0.49 E163 G 1.25 0.80 0.79 E163 H 1.32 0.82 0.69 E163 L 1.50 1.94 0.58 E163 N 0.91 1.00 0.77 E163 P 0.08 0.77 0.30 E163 R 1.12 0.49 0.72 E163 S 1.12 0.85 0.81 E163 V 1.13 0.55 0.69 E163 W 1.21 0.98 0.49 E163 Y 1.41 1.89 0.60 L164 A −0.14 −0.85 0.21 L164 C 0.09 0.91 0.63 L164 D −0.14 −0.85 0.12 L164 E −0.14 −0.48 0.18 L164 F 0.50 0.86 0.94 L164 G −0.14 −0.14 0.19 L164 H 0.02 0.12 0.16 L164 L 1.00 1.00 1.00 L164 M 0.69 1.26 1.09 L164 N −0.14 1.31 0.26 L164 P −0.14 2.41 0.17 L164 Q −0.14 1.01 0.24 L164 R −0.14 1.61 0.17 L164 S 0.32 1.11 0.25 L164 T 0.82 0.99 0.52 L164 V 0.87 1.02 1.08 L164 Y 0.43 −1.28 0.20 A165 A 1.00 1.00 1.00 A165 C 0.99 1.42 0.97 A165 D 0.89 1.69 0.62 A165 F 1.23 1.00 0.74 A165 G 1.05 1.07 1.14 A165 I 1.17 0.59 0.64 A165 K 1.35 0.82 0.78 A165 L 1.08 1.55 0.70 A165 M 0.97 1.56 0.77 A165 N 1.01 1.20 0.91 A165 P 1.14 1.34 0.91 A165 Q 1.21 1.32 1.05 A165 R 1.70 1.29 0.87 A165 S 1.00 0.94 1.05 A165 T 1.18 1.32 0.83 A165 V 1.21 1.13 0.88 A165 Y 1.20 0.84 0.67 R166 A 0.73 1.51 1.12 R166 D 0.56 1.55 1.16 R166 F 1.00 1.10 0.85 R166 G 1.15 0.91 1.19 R166 H 1.20 1.56 0.97 R166 I 1.26 1.39 0.86 R166 K 1.17 1.20 1.19 R166 L 1.27 1.50 1.08 R166 M 0.65 1.29 1.26 R166 N 0.75 1.21 1.16 R166 P 0.43 1.50 0.97 R166 R 1.00 1.00 1.00 R166 S 1.16 0.95 0.98 R166 T 1.19 0.74 1.04 R166 V 1.17 0.76 0.94 R166 W 1.25 1.08 0.80 R166 Y 1.29 1.22 0.85 V167 A 0.56 4.99 0.98 V167 C 0.79 5.37 1.01 V167 D 0.56 5.54 0.98 V167 G 0.99 2.83 1.08 V167 H 1.03 2.11 1.12 V167 I 1.08 1.00 1.04 V167 L 0.84 2.56 1.13 V167 M 0.53 3.84 1.04 V167 P 0.31 6.08 0.85 V167 Q 0.55 2.41 0.97 V167 R 0.78 2.25 0.88 V167 S 0.96 1.86 1.04 V167 T 1.13 2.47 0.96 V167 V 1.00 1.00 1.00 V167 Y 1.07 2.15 0.94 Y168 C 0.69 −4.73 0.57 Y168 D −0.11 −1.98 −0.03 Y168 E −0.11 −1.98 −0.03 Y168 F 0.68 5.17 1.28 Y168 G 1.89 −40.74 0.23 Y168 H −0.11 −1.98 −0.03 Y168 I 0.83 −0.59 0.90 Y168 K −0.11 −1.98 −0.03 Y168 L 0.59 5.39 1.27 Y168 N −0.11 −1.98 −0.03 Y168 P −0.11 −1.98 −0.03 Y168 Q 0.28 −8.27 0.25 Y168 R −0.11 −1.98 −0.03 Y168 S −0.11 −1.98 −0.03 Y168 T 1.51 −22.96 0.39 Y168 V 1.19 −12.96 0.57 Y168 W −0.11 −1.98 −0.03 Y168 Y 1.00 1.00 1.00 S169 A 0.94 1.13 0.95 S169 C 1.03 1.38 0.78 S169 I 1.16 1.53 0.66 S169 K 1.21 1.27 0.94 S169 L 1.08 1.47 0.82 S169 M 0.86 1.40 0.86 S169 P 0.87 0.89 0.69 S169 Q 1.02 1.37 0.88 S169 R 1.24 1.19 0.77 S169 S 1.00 1.00 1.00 S169 T 1.15 0.97 0.82 S169 Y 1.26 1.10 0.77 A170 A 1.00 1.00 1.00 A170 C 1.15 1.06 1.02 A170 D 1.27 1.32 0.88 A170 E 1.28 1.17 0.99 A170 F 1.44 1.17 0.83 A170 G 1.59 0.62 0.96 A170 I 1.59 0.44 0.95 A170 K 1.71 0.83 0.96 A170 L 1.05 0.85 0.87 A170 M 1.03 1.28 0.93 A170 N 1.21 1.17 0.96 A170 P 0.75 1.33 0.80 A170 Q 1.15 0.89 0.98 A170 S 1.47 0.47 0.99 A170 T 1.40 0.72 0.86 A170 V 1.20 0.74 0.83 A170 W 1.04 0.83 0.82 A170 Y 0.80 0.89 0.89 L171 A 0.35 1.66 0.79 L171 C 0.56 1.73 0.97 L171 D −0.06 −0.13 −0.01 L171 F 1.30 1.97 0.87 L171 G 1.26 1.33 0.50 L171 H 1.67 1.07 0.61 L171 I 1.53 1.42 1.16 L171 K 2.05 1.53 0.31 L171 L 1.00 1.00 1.00 L171 M 0.53 2.22 0.90 L171 N 0.96 2.79 0.40 L171 Q 0.97 1.93 0.67 L171 R 0.71 −0.20 0.24 L171 S 1.43 1.76 0.72 L171 T 1.54 1.36 0.80 L171 V 1.02 1.39 0.92 L171 Y 1.20 1.35 0.88 A172 A 1.00 1.00 1.00 A172 C 1.20 0.86 1.09 A172 D −0.15 1.42 0.16 A172 E −0.15 −0.44 0.19 A172 G 1.41 0.84 1.07 A172 I 1.70 0.58 0.30 A172 K 0.95 −0.43 0.17 A172 L 1.20 1.22 0.70 A172 M 0.84 1.06 0.84 A172 N 0.37 0.76 0.30 A172 P −0.15 0.58 0.16 A172 Q 0.27 0.18 0.34 A172 R 0.44 −0.18 0.20 A172 S 1.59 0.85 0.96 A172 T 1.25 0.71 0.85 A172 V 1.40 0.39 0.53 A172 W 1.43 0.45 0.12 A172 Y 0.87 1.76 0.13 S173 A 0.81 2.72 0.95 S173 C 0.82 3.07 0.59 S173 E 0.78 2.65 0.90 S173 F 0.96 2.30 0.71 S173 H 1.07 1.49 0.95 S173 I 0.99 2.22 0.78 S173 K 1.17 3.01 0.91 S173 L 1.15 3.86 0.77 S173 M 0.80 3.01 0.84 S173 P 0.19 2.66 0.35 S173 R 1.09 2.47 0.82 S173 S 1.00 1.00 1.00 S173 T 1.06 1.29 0.89 S173 V 0.95 2.54 0.75 S173 W 1.16 3.67 0.67 S173 Y 1.19 3.54 0.81 F174 A 0.59 2.09 0.61 F174 C 1.32 0.48 0.65 F174 F 1.00 1.00 1.00 F174 G 1.60 0.91 0.85 F174 H 0.93 1.05 0.86 F174 K 0.86 1.17 0.76 F174 L 1.05 1.83 0.82 F174 M 0.91 2.20 0.55 F174 P 1.54 1.46 0.13 F174 Q 1.42 0.46 0.82 F174 R 0.70 0.52 0.95 F174 S 1.16 0.61 0.75 F174 T 0.80 0.64 0.62 F174 V 0.60 0.67 0.82 F174 W 0.96 −0.02 0.85 F174 Y 0.84 1.66 0.77 M175 A 0.70 0.66 0.95 M175 E 0.95 1.43 0.89 M175 G 2.04 0.75 0.67 M175 L 1.61 0.86 1.19 M175 M 1.00 1.00 1.00 M175 N 1.39 1.02 1.11 M175 P −0.20 0.08 0.16 M175 Q 1.56 0.83 0.98 M175 R 1.55 0.86 1.02 M175 T 2.21 0.90 0.98 M175 V 1.93 0.81 1.00 M175 W 1.25 0.76 1.14 M175 Y 0.77 0.72 1.35 K176 A 0.42 1.19 0.84 K176 C 0.58 1.01 0.87 K176 D 0.62 1.18 0.74 K176 E 0.67 1.08 0.88 K176 F 0.36 1.28 0.31 K176 G 1.01 0.73 0.80 K176 K 1.00 1.00 1.00 K176 L 1.00 0.92 0.58 K176 M 0.56 1.33 0.74 K176 N 0.60 0.94 0.85 K176 P 0.01 0.78 0.27 K176 Q 0.59 0.97 1.02 K176 R 0.71 1.03 1.06 K176 S 0.76 0.72 0.93 K176 T 1.04 0.97 0.70 K176 V 1.04 1.33 0.71 K176 W 1.19 1.16 0.41 K176 Y 1.04 0.93 0.60 P178 A 0.31 4.39 0.96 P178 D 0.18 6.44 0.93 P178 E 0.40 4.15 1.05 P178 G 1.09 2.95 0.67 P178 K 1.34 1.70 0.73 P178 L 1.82 7.15 0.53 P178 M 0.53 3.87 0.78 P178 P 0.06 5.02 0.93 P178 Q 0.15 3.64 0.93 P178 S 0.62 3.06 0.95 P178 T 0.70 2.28 0.81 P178 V 0.67 2.70 0.78 P178 W 1.14 0.02 0.64 P178 Y 1.38 6.91 0.74 F179 A −0.18 −0.22 −0.02 F179 E 0.02 1.80 0.20 F179 F 1.00 1.00 1.00 F179 G 0.03 1.16 0.36 F179 H 0.79 0.93 0.91 F179 L 1.15 1.89 0.43 F179 N 0.77 0.95 0.46 F179 P −0.18 −0.22 −0.02 F179 Q 0.46 −0.87 0.46 F179 R −0.18 −0.22 −0.02 F179 S 0.78 0.34 0.62 F179 V 0.70 1.17 0.69 F179 W 0.89 0.86 0.62 F179 Y 1.05 1.47 0.65 F180 A 0.03 2.70 0.27 F180 C 0.65 1.94 0.66 F180 E −0.14 −0.55 −0.02 F180 F 1.00 1.00 1.00 F180 G 0.37 −5.96 0.20 F180 I 1.20 2.11 0.79 F180 K 1.08 −6.98 0.24 F180 L 1.30 2.13 0.86 F180 M 0.71 4.36 0.96 F180 N −0.14 3.05 0.29 F180 Q 0.21 −1.87 0.36 F180 R 0.64 −3.57 0.26 F180 S 0.56 −2.05 0.29 F180 T 1.01 −0.68 0.33 F180 V 1.14 3.24 0.76 F180 W 1.11 1.81 0.90 F180 Y 1.12 2.99 0.84 D181 A 1.35 1.23 0.65 D181 C 1.09 0.85 0.56 D181 D 1.00 1.00 1.00 D181 E 1.10 0.72 0.78 D181 F −0.15 −0.17 −0.01 D181 G 1.09 0.52 0.37 D181 H −0.15 −0.17 −0.01 D181 I −0.15 −0.17 −0.01 D181 K 1.33 0.47 0.41 D181 L 1.25 −0.16 0.16 D181 M −0.15 −0.17 −0.01 D181 N −0.15 −0.17 −0.01 D181 P 1.03 0.66 0.60 D181 Q 1.14 0.60 0.54 D181 R 1.23 0.22 0.45 D181 S 1.21 0.55 0.56 D181 T 1.02 −0.32 0.24 D181 V 0.88 −0.34 0.21 D181 W 1.26 −0.52 0.28 D181 Y 1.29 −0.25 0.25 A182 A 1.00 1.00 1.00 A182 C 0.97 0.99 1.03 A182 G 0.92 0.94 0.90 A182 H −0.14 −0.18 −0.02 A182 I 0.89 −2.48 0.20 A182 K −0.14 −0.18 −0.02 A182 L −0.14 −0.18 −0.02 A182 M −0.14 −0.18 −0.02 A182 N −0.14 0.53 0.14 A182 P −0.14 −1.13 0.12 A182 Q 0.03 −0.84 0.14 A182 R 0.25 −2.69 0.12 A182 S 0.87 0.85 0.90 A182 T 1.14 0.11 0.48 A182 W −0.14 −0.18 −0.02 A182 Y −0.14 −0.18 −0.02 G183 C 0.56 1.99 0.92 G183 D 0.30 0.99 0.62 G183 F 0.68 0.19 0.75 G183 G 1.00 1.00 1.00 G183 H 0.98 0.95 0.87 G183 L 0.82 1.50 0.47 G183 P −0.18 1.02 0.33 G183 Q 0.66 −0.20 0.97 G183 R 0.92 1.09 0.90 G183 S 0.94 −0.08 1.08 G183 V 0.56 −2.47 0.57 G183 Y 0.97 1.45 0.79 S184 A 0.60 1.69 1.31 S184 C 0.81 2.39 1.14 S184 D 0.84 2.24 1.15 S184 E 0.94 1.86 1.39 S184 F 1.05 1.27 0.89 S184 G 0.99 0.82 1.15 S184 H 1.02 0.74 1.07 S184 I 0.92 1.21 0.96 S184 K 0.97 1.61 1.02 S184 L 0.80 2.00 0.98 S184 M 0.51 1.77 1.25 S184 N 0.64 1.93 1.03 S184 P −0.15 0.85 0.40 S184 Q 0.89 1.16 1.09 S184 S 1.00 1.00 1.00 S184 T 1.04 0.60 0.94 S184 V 0.80 1.25 1.03 S184 Y 1.06 1.09 0.84 V185 C 0.65 0.83 0.96 V185 D 0.40 −2.49 0.21 V185 E 0.73 0.88 0.76 V185 F 1.02 1.20 0.83 V185 G 1.12 −3.67 0.47 V185 H 1.30 −0.58 0.71 V185 I 1.07 0.63 1.03 V185 K 1.37 0.79 0.66 V185 L 1.23 0.93 0.75 V185 M 0.39 1.46 0.77 V185 Q 0.77 1.41 0.73 V185 R 1.15 0.79 0.57 V185 S 1.09 0.53 0.75 V185 T 1.11 0.91 0.79 V185 V 1.00 1.00 1.00 V185 W 1.36 −0.44 0.53 V185 Y 1.37 0.58 0.65 I186 A 1.46 1.79 0.90 I186 D −0.13 4.29 0.19 I186 F 1.01 0.76 0.77 I186 G 1.86 −5.42 0.35 I186 I 1.00 1.00 1.00 I186 K −0.13 −0.36 −0.01 I186 L 1.17 1.14 0.84 I186 M 0.86 1.38 1.11 I186 P −0.13 −2.95 0.25 I186 R 0.62 −6.69 0.25 I186 S 1.39 −0.21 0.65 I186 T 1.51 0.23 0.79 I186 V 1.28 0.48 0.93 I186 W −0.13 −0.36 −0.01 I186 Y −0.13 −0.36 −0.01 S187 A 0.51 1.72 0.86 S187 C 0.70 1.67 0.79 S187 D 0.59 1.40 0.82 S187 F 1.02 0.65 0.73 S187 G 1.03 1.46 0.88 S187 H 1.29 1.51 0.68 S187 I 1.38 1.58 0.78 S187 K 1.45 1.16 0.76 S187 L 1.37 1.46 0.75 S187 M 0.49 1.87 0.85 S187 N 0.59 1.59 0.90 S187 P 0.44 −0.31 0.78 S187 Q 0.63 0.35 0.94 S187 R 1.04 0.55 0.82 S187 S 1.00 1.00 1.00 S187 T 1.12 0.23 0.74 S187 V 1.23 0.58 0.89 S187 W 1.30 0.52 0.73 S187 Y 1.43 0.80 0.76 T188 A 0.97 0.95 1.40 T188 C 0.60 0.87 2.04 T188 D −0.05 −0.14 −0.02 T188 E 0.24 1.97 0.44 T188 F 0.96 −0.20 0.63 T188 G 0.93 0.79 1.32 T188 H 1.11 −0.79 0.74 T188 I 1.13 0.10 1.85 T188 K −0.05 −0.14 −0.02 T188 L 0.76 0.42 1.76 T188 M 0.49 0.75 1.60 T188 N 0.69 1.69 1.24 T188 P −0.05 −0.14 −0.02 T188 Q −0.05 −0.14 −0.02 T188 R 1.01 −0.47 1.41 T188 S 1.16 0.91 1.52 T188 T 1.00 1.00 1.00 T188 V 1.22 0.15 1.53 T188 W −0.05 −0.14 −0.02 T188 Y 1.48 0.09 0.47 D189 A 0.05 1.18 0.53 D189 C 0.19 0.94 0.56 D189 D 0.03 0.89 0.90 D189 E 0.35 0.77 0.85 D189 F 0.83 0.37 0.63 D189 G 0.80 0.80 0.83 D189 H 1.25 0.95 0.78 D189 I 0.73 1.27 0.69 D189 L 1.30 1.30 0.61 D189 M 0.06 0.88 0.48 D189 N 0.22 0.57 0.80 D189 P −0.12 0.97 0.67 D189 R 0.86 0.39 0.65 D189 S 0.88 0.81 0.85 D189 T 1.00 1.21 0.73 D189 V 0.73 0.71 0.72 D189 W 1.09 0.76 0.60 I194 A 0.29 0.00 1.15 I194 C 0.27 −0.02 1.17 I194 F 0.07 −0.03 0.95 I194 G 0.10 0.04 0.34 I194 I 1.00 1.00 1.00 I194 L 0.80 0.58 1.32 I194 P 0.15 −1.42 0.16 I194 R 0.02 −0.40 0.77 I194 S 0.30 −0.15 0.48 I194 V 0.37 0.78 1.03 I194 W 0.04 −0.09 1.12 I194 Y −0.32 −0.01 1.01 F196 A −0.13 −0.13 −0.02 F196 C 1.74 1.18 0.70 F196 F 1.00 1.00 1.00 F196 G 1.59 −0.30 0.60 F196 H 1.77 −0.24 0.23 F196 I 1.32 1.12 0.81 F196 K −0.13 −0.13 −0.02 F196 L 1.77 1.17 1.09 F196 M 1.65 0.71 0.93 F196 N −0.13 −0.13 −0.02 F196 P 0.05 0.39 0.42 F196 Q 1.00 −0.25 0.40 F196 R −0.13 −0.13 −0.02 F196 S 1.58 −1.57 0.29 F196 V 1.40 0.68 0.51 F196 W 1.01 0.38 0.88 F196 Y 1.41 0.97 0.73

Example 11 Cloning and Expression of a Sinorhizobium meliloti RSM02162 M. smegmatis Perhydrolase Homologue

In this Example, cloning and expression of a S. meliloti perhydrolase homologue are described. The sequences used in cloning and expression are provided below. The gene RSM02162 (SEQ ID NO:625) was synthesized by DNA2.0. The gene was given the designation “G00355” and was provided cloned into the commercially available vector, pDRIVE (InvivoGen). The gene was amplified by PCR from this clone using the primer set G00355rbsF G00355R, Taq DNA polymerase (Roche) as per the manufacturer's directions, with G00355 as the template (10 ng/50 μl reaction) and 10 picomoles (per 50 μl reaction) of each primer. The amplification was carried out in an MJ Research PCR machine using 30 cycles of (1 minute at 95° C.; 1 minute at 55° C.; and 1 minute at 72° C.). The amplification of the correct size fragment was confirmed by agarose gel electrophoresis. The fragment was cloned directly into pCR2.1TOPO (Invitrogen) and transformed into E. coli Top10 cells (Invitrogen). Transformants were selected on L agar containing carbenicillin (100 μg/ml) at 37° C. The correct construct was confirmed by sequence analysis and designated “pMC355rbs.” FIG. 20 provides a map of this plasmid.

Primer sequences: G00355rbsF (SEQ ID NO: 626) 5′-ggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgc-3′ G00355R (SEQ ID NO: 627) 5′-Gcgcgcttagaacagagccgctactttgtcagc-3′ Gene sequence (including stop codon) of RSM02162: (SEQ ID NO: 625) 5′- atggtggaaaaacgttccgttctgtgctttggtgattctctgacttggggctggattccggtgaaagagagctccccaactctgcgtt acccatacgaacagcgttggaccggtgctatggctgcacgtctgggtgatggttaccacatcattgaagaaggcctgtccgctcgt actactagcctggacgacccaaacgacgctcgtctgaacggctctacctacctgccgatggctctggcttcacctgccactgga tctggtaatcattatgctgggtaccaacgacaccaaaagctactttcatcgtaccccatacgagattgccaacggcatgggtaaact ggtaggtcaggtcctgacctgtgcaggtggtgttggtacgccttatccagcaccgaaagtcctggtggttgcacctccaccactgg caccaatgccagatccgtggttcgaaggtatgttcggcggtggttacgagaaatctaaggaactgtccggtctgtacaaagcactg gctgatttcatgaaagtggagttcttcgcagcgggtgattgtatctccaccgacggtatcgacggtatccacctgagcgctgaaacc aacatccgcctgggtcatgctattgctgacaaagtagcggctctgttctaa-3′ G00355 Protein sequence: (SEQ ID NO: 628) MVEKRSVLCFGDSLTWGWIPVKESSPTLRYPYEQRWTGAMAARLGDGYHIIEEG LSARTTSLDDPNDARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIA NGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAPMPDPWFEGMFGGGYEKS KELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF Complete sequence of pDRIVEG00355: (SEQ ID NO: 629) gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagc gggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttg tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctctaatacgactcactataggg aaagctcggtaccacgcatgctgcagacgcgttacgtatcggatccagaattcgtgattttagaacagagccgctactttgtcagca atagcatgacccaggcggatgttggtttcagcgctcaggtggataccgtcgataccgtcggtggagatacaatcacccgctgcga agaactccactttcatgaaatcagccagtgctttgtacagaccggacagttccttagatttctcgtaaccaccgccgaacataccttc gaaccacggatctggcattggtgccagtggtggaggtgcaaccaccaggactttcggtgctggataaggcgtaccaacaccacc tgcacaggtcaggacctgacctaccagtttacccatgccgttggcaatctcgtatggggtacgatgaaagtagcttttggtgtcgttg gtacccagcataatgattaccagatccagtggcaggtgagaagccagagccatcggcaggtaggtagagccgttcagacgagc gtcgtttgggtcgtccaggctagtagtacgagcggacaggccttcttcaatgatgtggtaaccatcacccagacgtgcagccatag caccggtccaacgctgttcgtatgggtaacgcagagttggggagctctctttcaccggaatccagccccaagtcagagaatcacc aaagcacagaacggaacgtttttccaccataatctgaattcgtcgacaagcttctcgagcctaggctagctctagaccacacgtgtg ggggcccgagctcgcggccgctgtattctatagtgtcacctaaatggccgcacaattcactggccgtcgttttacaacgtcgtgact gggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatggaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgt taaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttg ttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggccc actacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgat ttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctg gcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcaggtggcactttt cggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttt gctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcc cgtattgacgccgggcaagagcaactcggtcgccgcatacactaactcagaatgacttggttgagtactcaccagtcacagaaaa gcatcaacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacactgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggccctt ccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaa gccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcc tcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatcctttttgataatctcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaac gggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggc aatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaat gatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatga tgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgagat gcgctggcagtgttcctgcgccggagcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcg caatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaa atgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacagataaccttatttttgacgaggggaaattaat aggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctcct tcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaa gaattaattcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttga gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccactt caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttacc gggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgga gcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcc tgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacg cggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccg cctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaaga Complete sequence pMC355rbs: (SEQ ID NO: 630) agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaag cgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgtt gtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggtaccgagctcggatcca ctagtaacggccgccagtgtgctggaattcgcccttggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgc tttggtgattctctgacttggggctggattccggtgaaagagagctccccaactctgcgttacccatacgaacagcgttggaccggt gctatggctgcacgtctgggtgatggttaccacatcattgaagaaggcctgtccgctcgtactactagcctggacgacccaaacga cgctcgtctgaacggctctacctacctgccgatggctctggcactcacctgccactggatctggtaatcattatgctgggtaccaac gacaccaaaagctactttcatcgtaccccatacgagattgccaacggcatgggtaaactggtaggtcaggtcctgacctgtgcag gtggtgttggtacgccttatccagcaccgaaagtcctggtggttgcacctccaccactggcaccaatgccagatccgtggttcgaa ggtatgttcggcggtggttacgagaaatctaaggaactgtccggtctgtacaaagcactggctgatttcatgaaagtggagttcttc gcagcgggtgattgtatctccaccgacggtatcgacggtatccacctgagcgctgaaaccaacatccgcctgggtcatgctattgc tgacaaagtagcggctctgttctaagcgcgcaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatct agagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtt acccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaac agttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga ccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagct ctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgta gtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaaca acactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaa atttaacgcgaattttaacaaaattcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacg gtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgca gtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggta aggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaaga gacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggct atgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaag accgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgc agctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatccca ccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgacc accaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc aggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcga tgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatca ggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttcc gtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatc agttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaa tgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctg ccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatg gaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatata tactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtg agttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca gagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcg ctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac aggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag Expression of the Homologue from pMC355rbs

To express the S. meliloti RSM02162 protein from the plasmid pMC355rbs (See, FIG. 20, for a map of this plasmid), a single colony was inoculated into a 5 mls of L broth containing 100 μg/ml carbenicillin and grown overnight at 37° C. with shaking at 200 rpm. Lysates were prepared by pelleting the cells from 1 ml of the overnight culture by centrifugation and lysed with BugBuster (Novagen). The supernatants were assayed using the pNA activity assay, perhydrolysis assay, and a pNC6 assay (to test its ability to hydrolyze carbon chains longer than C4), as described herein.

Assay Results

The following Table (Table 11-1) provides a comparison of the hydrolysis activity of pNA by G00355 as compared to the M. smegmatis perhydrolase

TABLE 11-1 pNA Hydrolysis Activity pNA Hydrolysis Rate Compared to Strain Rate* Perhydrolase E. coli/pMSATNcoI 85 1 E. coli/pMC355rbs 80 0.94 E. coli/pCR2.1 34.6 0.41 *Rate is absorbance units/min read at 405 nm in a spectrophotometer.

*Rate is absorbance units/min read at 405 nm in a spectrophotometer.

The following Table (Table 11-2) provides a comparison of the perhydrolysis of triacetin by G00355 compared to the M. smegmatis perhydrolase.

TABLE 11-2 Triacetin Perhydrolysis Activity Perhydrolysis Activity Strain Max Vmax E. coli/pMSATNcoI 1.04 11.88 E. coli/pMC355rbs 1.17 25.05 E. coli/pCR2.l 0.1 2.9

The following Table (Table 11-3) provides a comparison of pNC6 hydrolysis by G00355 compared to the M. smegmatis perhydrolase.

TABLE 11-3 pNC6 Hydrolysis Activity pNC6 Hydrolysis Rate Compared to Strain Rate* Ms. Perhydrolase E. coli/pMSATNcoI 0.58 1 E. coli/pMC355rbs 6.57 11.3 E. coli/pCR2.l 0.47 0.8 *Rate is absorbance units/min read at 405 nm in a spectrophotometer.

* Rate is absorbance units/min read at 405 nm in a spectrophotometer.

As these results indicate, the homologue RSM02162 from S. meliloti identified by amino acid sequence homology to the M. smegmatis perhydrolase demonstrated similar, albeit less perhydrolysis activity than the M. smegmatis perhydrolase. However, this enzyme exhibited different substrate specificity, as it was able to hydrolyze pNC6, while the wild-type M. smegmatis perhydrolase cannot.

The results of the pNC6 hydrolysis assay indicated that certain positions/substitutions provided an improvement in the ability of the enzyme to utilize longer chain substrates The positions and substitutions identified in preliminary screens are provided in the following Table. It is not intended that the present invention be limited to these specific positions and substitutions, as it is contemplated that additional positions and/or substitutions will also provide improved activity on longer chain substrates.

TABLE 11-4 Positions/Substitutions with Improved Activity in PNC6 Assay Wild-Type Residue/Position Amino Acid Variant(s) L12 G, P, Q S54 L, T I153 F, P F154 Q, S, T, V I194 G F196 A, C, G, I, N, P, Q, S, V

Example 12 Amplification of Genes Encoding M. smegmatis Perhydrolase Homologues from Environmental Isolates

In this Example, methods used to amplify genes encoding M. smegmatis perhydrolase homologues from environmental isolates are described.

Organisms from soil samples that were positive for the transesterification reaction were purified to single colonies. To amplify the genes by PCR, the degenerate primer sets 1AF5AR and 1eF51R were used in a PCR reaction containing isolated chromosomal DNA from 8 environmental strains exhibiting the transesterification reaction. The PCR reaction was carried out using Taq DNA polymerase (Roche) as per the manufacturer's protocol, with 1 μg of chromosomal DNA added as template and 10 picomoles of each primer in a 50 μl reaction. The reaction was carried out for 30 cycles of (1 minute at 95° C.; 1 minute at 50° C., and 1 minute at 72° C.). Since the partial coding sequence of the perhydrolase gene from Mycobacterium parafortuitum was already isolated, the same strain was used as a positive control. The strains were designated as: 2G, 2D, 9B, 14B, 18D, 19C, 20A. As indicated below, 20A was typed as Mycobacterium parafortuitum, and 9B is Mycobacterium gilvum. Based on protein homology, it was inferred that 2D is also M. parafortuitum and 14B is M. gilvum.

Primer Sequences

1AF: (SEQ ID NO: 631) 5′-gccaagcgaattctgtgtttcggngaytcnyt-3′ 5AR: (SEQ ID NO: 632) 5′-cgattgttcgcctcgtgtgaartgnrtnccrtc-3′ 1eF: (SEQ ID NO: 633) 5′-acggtcctgtgctttggngaytcnyt-3′ 5iR: (SEQ ID NO: 634) 5′-ccgctggtcctcatctggrtgntcnccrtc-3′

Amplification with the above primer sets was expected to yield bands of approximately 500 bp. In all cases except 2G, the 1AF/5AR primer set produced a band of the expected size. In the case of 19C, both primer sets produced bands of the expected size. The 500 bp bands were purified from agarose gels using a gel purification kit (Qiagen) and analyzed by sequencing. While the strains 2G and 19C yielded bands of the expected size with both primer sets they were not the fragments encoding the M. smegmatis perhydrolase homologue.

Partial Sequences of 2D Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 635) 5′-attctgtgtttcggggattccttgacgtggggatggatccctgtcga agaaggtgtgcccaccgagcggttcccgcgtgacgtccggtggaccggcg tgctggccgacctgctgggcgaccgctacgaggtgatcgaggaaggcctg tcggcgcgcaccaccaccgccgacgacccggccgacccccggctcaacgg ttcgcagtatctgccgtcgtgtctggccagccatctgccgctggacctgg tgatcctgatgctcggcatcaacgacaccaaggcgaattttggccgcacc ccgttcgacatcgccaccggtatgggagtgcttgccacgcaggtgctcac cagcgccggtggcgtggggaccagctatcccgcgccgcaggtgctgatcg tggcgccgccgccgctgggcgagctgccccacccctggttcgacctggtg ttctccggcggccgtgagaagaccgccgagttggcccgcgtgtacagcgc gctggcgtcgttcatgaaggtgccgttcttcgacgccggctcggtgatca gcaccgacggcgtggacggcacccacttcacacgaggcgaaacaatcga Protein: (SEQ ID NO: 636) ILCFGDSLTWGWIPVEEGVPTERFPRDVRWTGVLADLLGDRYEVIEEGLS ARTTTADDPADPRLNGSQYLPSCLASHLPLDLVILMLGINDTKANFGRTP FDIATGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWFDLVF SGGREKTAELARVYSALASFMKVPFFDAGSVISTDGVDGTHFTRGETI

Partial Sequences of 9B Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 637) 5′-taccgtcgatgtgtggcctcgtgtgaagtgggtgccgttgccaagcg aattctgtgtttcggggattcgttgacgtggggctggatcccggtcgagg aaggtgtacccacccaacgttttccgaagcgggtgcgctggaccggggtg ctggccgacgaactgggtgctggctatgaggttgtcgaggaggggttgag cgcgcgcaccaccaccgctgacgaccctaccgatccccggctgaacggct cggactacctccccgcatgcctggccagccacctgccgctggacctggtg atcctgatgctcgggaccaacgacaccaaggcgaatctgaatcgcacacc cgtcgacatcgccagcggaatgggcgtcctggccacccaggtgctcacca gcgcgggcggggtcggcaccagctacccggccccgcaggtgttgatcgtg gcaccgccgccgctggccgagatgccgcacccgtggttcgagctggtctt cgacggcggccgggagaagaccgcccaactggcccgggtgtacagcgcgc tggcgtcgttcatgaaggtgccgttcttcgacgccggatcggtgatcagc accgacggtgtcgacggcacccacttcacacgaggcgaaacaatcgac cgg Protein: (SEQ ID NO: 638) GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFPKRVRWTGVL ADELGAGYEVVEEGLSARTTTADDPTDPRLNGSDYLPACLASHLPLDLVI LMLGTNDTKANLNRTPVDIASGMGVLATQVLTSAGGVGTSYPAPQVLIVA PPPLAEMPHPWFELVFDGGREKTAQLARVYSALASFMKVPFFDAGSVIST DGVDGTHFTRGETIDR

Partial Sequences of 14B Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 639) 5′-attctgtgtttcggagattcgttgacgtggggctggatcccggtcga ggaaggtgtacccacccaacgttttccgaagcgggtgcgctggaccgggg tgctggccgacgaactgggtgctggctatgaggttgtcgaggaggggttg agcgcgcgcaccaccaccgctgacgaccctaccgatccccggctgaacgg ctcggactacctccccgcatgcctggccagccacctgccgctggacctgg tgatcctgatgctcgggaccaacgacaccaaggcgaatctgaatcgcaca cccgtcgacatcgccagcggaatgggcgtcctggccacccaggtgctcac cagcgcgggcggggtcggcaccagctacccggccccgcaggtgttgatcg tggcaccgccgccgctggccgagatgccgcacccgtggttcgagctggtc ttcgacggcggccgggagaagaccgcccaactggcccgggtgtacagcgc gctggcgtcgttcatgaaggtgccgttcttcgacgccggatcggtgatca gcaccgacggtgtcgacggcacccacttcacacgagg Protein: (SEQ ID NO: 640) ILCFGDSLTWGWIPVEEGVPTQRFPKRVRWTGVLADELGAGYEVVEEGLS ARTTTADDPTDPRLNGSDYLPACLASHLPLDLVILMLGTNDTKANLNRTP VDIASGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLAEMPHPWFELVF DGGREKTAQLARVYSALASFMKVPFFDAGSVISTDGVDGTHFTR

Partial Sequences of 20A Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 641) 5′-ttgccaagcggaattctgtgtttcggggattctttgacgtggggatg gatccctgtcgaagaaggtgtgcccaccgagcggttcccgcgtgacgtcc ggtggaccggcgtgctggccgacctgctgggcgaccgctacgaggtgatc gaggaaggcctgtcggcgcgcaccaccaccgccgacgacccggccgaccc ccggctcaacggttcgcagtatctgccgtcgtgtctggccagccatctgc cgctggacctggtgatcctgatgctcggcatcaacgacaccaaggcgaat tttggccgcaccccgttcgacatcgccaccggtatgggagtgcttgccac gcaggtgctcaccagcgccggtggcgtggggaccagctatcccgcgccgc aggtgctgatcgtggcgccgccgccgctgggcgagctgccccacccctgg ttcgacctggtgttctccggcggccgtgagaagaccgccgagttggcccg cgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccg gctcggtgatcagcaccgacggcgtggacggcacccacttcacacgaggc gaaacaatcga-3′ Protein: (SEQ ID NO: 642) LPSGILCFGDSLTWGWIPVEEGVPTERFPRDVRWTGVLADLLGDRYEVIE EGLSARTTTADDPADPRLNGSQYLPSCLASHLPLDLVILMLGINDTKANF GRTPFDIATGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWF DLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDGVDGTHFTRG ETI

Identification of the Natural Isolates

To type the environmental isolates used in this Example, plates of the purified strains were sent to MIDI for 16S rRNA typing. 20A is Mycobacterium parafortuitum, 9B is Mycobacterium gilvum. By protein homology we infer that 2D is also M. parafortuitum and 14B is M. gilvum.

Example 13 Sequence and Taxonomic Analyses of Perhydrolase Homologues

In this Example, sequence and taxonomic analyses of M. smegmatis perhydrolase homologues are provided

Taxonomic Assignment

The basic “List of 60” protein sequences accessed from public databases and used for construction of primer sets for screening of metagenomic libraries (BRAIN) was converted into a document illustrating the microbial taxonomic origins of the proteins, as described below. This information was used to produce the following alignment.

1                                               50 MSAT (1) -------------MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG 14B natural isolate (1) -----------------ILCFGDSLTWGWIPVEEGVPT-QRFPKRVRWTG 20A (1) -------------LPSGILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG 2D natural isolate (1) -----------------ILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG 9B Natural Isolate (1) -GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPT-QRFPKRVRWTG M. parafortuitum CO1 (1) -------------MAKRILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG Sm-RSM05666 (1) --------------MKTVLCYGDSLTWGYDATGSG-----RHALEDRWPS At-Q8UAC0 (1) --------------MKTVLAFGDSLTWGADPATG---L--RHPVEHRWPD At-Q8UFG4 (1) -------------MVKSVLCFGDSLTWGSNAETGG-----RHSHDDLWPS M091_M4aE11 (1) --------------MKTILAYGDSLTYGANPIPGGP----RHAYEDRWPT M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGG------RHALEDRWPS P.dejongeii RVM04532 (1) --------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhizobium meliloti (1) ---------MEETVARTVLCFGDSNTHGQVPGRGPLDR---YRREQRWGG Q98MY5 Mesorhizobium loti (1) --------------MKTVLCYGDSLTWGYNAEGG------RHALEDRWPS RSM02162_Sm (1) -----------MVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG S261_M2aA12 (1) --------------MKNILAFGDSLTWGFVAGQDAR-----HPFETRWPN Sma1993 Sinorhizobium (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPPEQRWTG meliloti Consensus (1)                KTILCFGDSLTWGWIPV EG P   RHP E RW G 51                                             100 MSAT (37) VLAQQLGADFEVIE--EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP 14B natural isolate (33) VLADELGAGYEVVE--EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP 20A (37) VLADLLGDRYEVIE--EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 2D natural isolate (33) VLADLLGDRYEVIE--EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 9B Natural Isolate (49) VLADELGAGYEVVE--EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP M. parafortuitum CO1 (37) VLADLLGDRYEVIE--EGLSARTTTAEDPADPRL-NGSQYLPSCLASHLP Sm-RSM05666 (32) VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP At-Q8UAC0 (32) VLEAELAGKAKVHP--EGLGGRTTCYDDHAGPACANGARALEVALSCHMP At-Q8UFG4 (33) VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M091_M4aE11 (33) ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADANGARVLPTLLESHSP M1-RML000301 (45) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADANGARLLPTALTTHAP P.dejongeii RVM04532 (37) VLAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE--EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP Q98MY5 Mesorhizobium loti (31) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADANGARLLPTALTTHAP RSM02162_Sm (39) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP S261_M2aA12 (32) ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP Sma1993 Sinorhizobium (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP meliloti Consensus (51) VLA  LGG Y VIE  EGLSGRTT  DDP D  L NGS YLPT  LASHLP 101                                            150 MSAT (84) LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLVUQVLUSAGGVGUUYPA 14B natural isolate (80) LDLVILMLGTNDTKANLNRTPVDIA--SGMGVLATQVLTSAGGVGTSYPA 20A (84) LDLVILMLGINDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA 2D natural isolate (80) LDLVILMLGINDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA 9B Natural Isolate (96) LDLVILMLGTNDTKANLNATPVDIA--SGMGVLATQVLTSAGGVGTSYPA M. parafortuitum CO1 (84) LDLVILMLGTNDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA Sm-RSM05666 (80) LDLIVFMLGSNDMKPIIHGTAFGAV--KGIERLVNLVRAHDWPTETE-EG At-Q8UAC0 (80) LDLVIIMLGINDIKPVHGGRAEAAV--SGMARLAQIVETFIYKPREA--V At-Q8UFG4 (83) LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDW-EA M091_M4aE11 (81) LDLIVIMLGTNDIKPHHGRTAGEAG--RGMARLVQIIRGHYAGRMQD--E M1-RML000301 (93) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWP--A P.dejongeii RVM04532 (84) LDLVILMLGTNDLKSTFNVPPGEIA--AGAGVLGRMILAGDAGPENR--P Q92XZ1 Sinorhizobium meliloti (87) LDLIIIMLGTNDLKARFNMPPSEVA--MGIGCLVHDIRELSPGRTGN--D Q98MY5 Mesorhizobium loti (79) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWP--A RSM02162_Sm (86) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGLPYPA S261_M2aA12 (80) LDLVIIMLGTNDIKFAARCRAFDAS--MGMERLIQIVRSANYMKGYK--I Sma1993 Sinorhizobium (97) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA meliloti Consensus (101) LDLVIIMLGTNDMKA   RTP DIA   GMGRLV  VLT AGGVG    A 151                                            200 MSAT (132) PKVLVVSPPPLAPM-PHPWFQLIF-EGGEQKUUELARVYSALASFMKVPF 14B natural isolate (128) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF 20A (132) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF 2D natural isolate (128) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF 9B Natural Isolate (144) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF M. parafortuitum CO1 (132) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF Sm-RSM05666 (127) PEILIVSPPPLCET--ANSAFAAMFAGGVEQSAMLAPLYRDLADELDCGF At-Q8UAC0 (126) PKLLIVAPPPCVAG---PGGEPAG-GRDIEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (132) PDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M091_M4aE11 (127) PQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF Ml-RMLO00301 (139) PQILIVSPPVVSRT--ENADFREMFAGGDEASKQLAPQYAALADEVGCGF P. dejongeii RVM04532 (130) PQLLLMCPPKVRDLSAMPDLDAKI-PHGAARSAEFPRHYKAQAVALKCEY Q92XZ1 Sinorhizobium meliloti (133) PEIMIVAPPPMLED--LKEWESIF-SGAQEKSRKLALEFEIMADSLEAHF Q98MY5 Mesorhizobium loti (125) PQILIVSPPVVSRT--ENADFREMFAGGDEASKQLAPQYAALADEVGCGF RSM02162_Sm (134) PKVLVVAPPPLAPM-PDPWFEGMF-GGGYEKSKELSGLYKALADFMKVEF S261_M2aA12 (126) PEILIISPPSLVPT--QDEWFNDLWGHAIAESKLFAKHYKRVAEELKVHF Sma1993 Sinorhizobium (145) PKVLVVAPPPLAPM-PDPWFEGMF-GGGYEKSKELSGLYKALADFMKVEF meliloti Consensus (151) PQVLIVAPPPL EM   P FE VF  GG EKS  LARVY ALAD MKV F 201                                   241 MSAT (180) FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL---- (SEQ ID NO: 643) 14B natural isolate (176) FDAGSVISTDGVDGTHFIR---------------------- (SEQ ID NO: 644) 20A (180) FDAGSVISTDGVDGTHFTRGETI------------------ (SEQ ID NO: 645) 2D natural isolate (176) FDAGSVISTDGVDGTHFTRGETI------------------ (SEQ ID NO: 646) 9B Natural Isolate (192) FDAGSVISTDGVDGTHFTRGETIDR---------------- (SEQ ID NO: 647) M. parafortuitum CO1 (180) FDAGSVISTDGVDGIHFTRGEQST----------------- (SEQ ID NO: 648) Sm-RSM05666 (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPVVRMMLGL-- (SEQ ID NO: 649) At-Q8UAC0 (172) FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG--- (SEQ ID NO: 650) At-Q8UFG4 (182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL-- (SEQ ID NO: 651) M091_M4aE11 (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL-- (SEQ ID NO: 652) Ml-RML000301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML---- (SEQ ID NO: 653) P. dejongeii RVM04532 (179) FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG--- (SEQ ID NO: 654) Q92XZ1 Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO: 655) Q98MY5 Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVMLEL-- (SEQ ID NO: 656) RSM02162_Sm (182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 657) S261_M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL-- (SEQ ID NO: 658) Sma1993 Sinorhizobium (193) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 659) meliloti Consensus (201) FDAGSVISTD VDGIHLDA  T  IG AL   VR LL (SEQ ID NO: 660)

The alignment tree from the CLUSTALW alignment (which approximates to a phylogenetic tree) suggests 3 or 4 groupings.

From this alignment, a hypothetical protein sequence was constructed from the consensus sequence. Where no consensus existed the site was filled with the Per amino acid; gaps were ignored. This provided a Per-consensus sequence:

(SEQ ID NO: 661) 1 TILCFGDSLT WGWIPVEEGA PTERHPPEVR WTGVLAQQLG GDYEVIEEGL 51 SGRTTNIDDP TDPRLNGSSY LPTCLASHLP LDLVIIMLGT NDMKAYFRRT 101 PLDIALGMGR LVTQVLTSAG GVGTTYPAPQ VLIVAPPPLA EMPHPWFELV 151 FEGGEEKSTE LARVYSALAD FMKVPFFDAG SVISTDGVDG IHLDAANTRD 201 IGVALAEQVR SLL

This consensus sequence was used for a BLASTP search against a non-redundant database. This search identified 55 hits. The majority of the ‘hits’ were GDSL or GDSI type molecules covering a wide range of microbial diversity. However, only the first 14 ‘hits’ had e-values and bit-values in the reliable range. At first sight, this appeared to provide further molecules with a GDSL/N-G/ARTT motif, but this was found to be due to differences in coding (Swiss Prot vs GenBank)

The screening of 3 environmental libraries (at BRAIN) resulted in 10 clones with a GDSL motif. A further 2 clones were derived from the BRAIN library. The following Table (Table 13-1) lists the clones and indicates their activity.

TABLE 13-1 Clones with GDSL Motifs Library Clone Perhydrolase Activity S248Fa S248_M40cD4 No S248Fa S248_M44aA5 No S248Fa S248_M18bH12 Not Perhydrolase S248Fa S248_M36bC5 Not Perhydrolase S248Fa S248_M50cD9 Not Perhydrolase S248Fa S248_M2bB11 ? Low S261 S261_M2aA12 Yes S279 S279_M75bA2 Not done S279 S279_M11aC12 Not GDSL S279 S279_M70aE8 ? Low M091 M091_M4aE11 Not tested BRAIN Est114 No BRAIN Est105 Not done

M40cD4

Strongest hit: arylesterase of Brucella melitensis (46% identical). Motifs: GDSL-GAND; GQTT instead of GRTT. Sequence alignment against the core list of organisms places it close to Caulobacter vibrioides and Brucella melitensis in the alpha-Proteobacteria.

M44aA5

Strongest hit:Acyl-CoA thioesterase of Pseudomonas aeruginosa (43% identical). Motifs: GDSL GGND; no GRTT or equivalent. Sequence alignment against the core list of organisms places it close to Pseudomonas sp in the gamma-Proteobacteria.

M2bB 11

Strongest hit: arylesterase of Brucella melitensis. Motifs: GDSL GAND; no

GRTT or equivalent. Sequence alignment against the core list of organisms shows no strong association placing it between the alpha- and gamma-Proteobacteria.

M2aA12

Strongest hit: arylesterase of Agrobacterium tumefaciens (42% identical) Motifs: GDSL-GRTT-GTND. Sequence alignment against the core list of organisms places it close to Agrobacterium tumefaciens in the alpha-Proteobacteria.

M75bA2

Strongest hit: incomplete. BLAST search revealed nothing significant. Motifs: GDSL-GTND; no GRTT or equivalent. Sequence alignment against the core list of organisms shows no convincing associations. The closest neighbors appear to be the Vibrio-Aeromonas groups of the gamma-Proteobacteria.

M70aE8

Strongest hit: acyl-CoA thioesterase from E. coli (30% identical), and aryl esterase hydrolase from Vibrio mimicus (27% identical). Based on incomplete sequence GDSL-type esterase (BRAIN) from Neisseria meningitidis (50% identical). Motifs: GDSL-GGND; no GRTT—replaced with GRTV. Sequence alignment against the core list of organisms shows the closest association to Neisseria meningitidis, a member of the beta-Proteobacteria.

M4aE11

Strongest hit: arylesterase from Agrobacterium tumefaciens (59% identity) Motifs: GDSL-GRTT-GTND. Sequence alignment against the core list of organisms shows the closest association to members of the alpha-Proteobacteria such as Agrobacterium.

Est114

Strongest hit: phosphatidylcholine sterol acyltransferase from Aeromonas hydrophila (gamma-Proteobacteria) (30% identical). Motifs: GDSL-GPND; no GRTT but GATT may be an equivalent. Sequence alignment against the core list of organisms shows the closest association to Acidophilium sp. and Aeromonas/Vibrio within the gamma-Proteobacteria.

Est105

Strongest hit: Pseudomonas aeruginosa outer membrane esterase, and hypothetical protein Pseudomonas putida (27% identical). Motifs: GDSL-GAND, no GRTT or equivalent. Sequence alignment against the core list of organisms shows the closest association to members of the gamma-Proteobacteria.

An overall alignment of these clones/sequences (here shown underlined) indicates that they are scattered throughout the alignment tree of strains indicating that the metagenomic screening has provided a variety of sequences and not a limited diversity.

Gene Mining for GRTT-Type Esterases

(clones with perhydrolase activity)

Sinorhizobium meliloti Sma1993-hypothetical protein_Sme

Motifs: GDSL-ARTT-GTND

Sinorhizobium meliloti Q92XZ1-hypothetical protein_Sme

Motifs: GDSN-GRTT-GTND

Mesorhizobium loti Q98MY5-arylesterase_Mlo

Motifs:GDSL-GRTT-GAND

Moraxella bovis AAK53448 (lipase) Motifs: GDSL-GSND, no GRTT or equivalent in this sequence order. (perhydrolase activity low, questionable sequence) Agrobacterium tumefaciens Q8UACO

Motifs: GDSL-GRTT-GTND

Agrobacterium tumefaciens Q8UFG4

Motifs: GDSL-GRTT-GTND

Mesorhizobium loti RML000301

Motifs: GDSL-GRTT-GAND

Sinorhizobium meliloti RSM05666

Motifs: GDSL-GRTT-GSND

(this clone was inactive for perhydrolase activity; and probably represents a false negative) Sinorhizobium meliloti RSM02162

Motifs: GDSL-ARTT-GTND

Prosthecobacter dejongeii RVM05432

Motifs: GDSN-GRTT-GTND

A GDSx₁-x₂RTT-Gx₃ND motif characterizes the active clones/sequences,

where:

X₁=L or N

X₂=A or G

X₃=T or A or S

The Moraxella bovis AAK53448 sequence does not fit this pattern and is excluded from the alignment analysis provided below:

Multiple Sequence Alignment of Active Clones/Sequences

Multiple Sequence Alignment of Active Clones/Sequences 1                                               50 ACT MSMEG (1) -------------MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG Q98MY5 Mesorhizobium loti (1) --------------MKTVLCYGDSLTWGYNAEGGR------HALEDRWPS Sma1993 Sinorhizobium (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG meliloti Q92XZ1 Sinorhizobium meliloti (1) ---------MEETVARTVLCFGDSNTHGQVPGRGPLDR---YRREQRWGG P. dejongeii RVM04532 (1) --------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG RSM05666_Sm (1) --------------MKTVLCYGDSLTWGYDATGSG-----RHALEDRWPS RSM02162_Sm (1) -----------MVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG At-Q8UAC0 (1) --------------MKTVLAFGDSLTWGADPATGLR-----HPVEHRWPD At-Q8UFG4 (1) -------------MVKSVLCFGDSLTWGSNAETGG-----RHSHDDLWPS M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGGR------HALEDRWPS S261_M2aA12 (1) --------------MKNILAFGDSLTWGFVAGQDA-----RHPFETRWPN M091_M4aE11 (1) --------------MKTILAYGDSLTYGANPIPGG-PR---HAYEDRWPT Consensus (1)               MKTVLCFGDSLTWGY P  G      RHA E RWP 51                                             100 ACT MSMEG (37) VLAQQLGADFEVIE--EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP Q98MY5 Mesorhizobium loti (31) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP Sma1993 Sinorhizobium (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP meliloti Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE--EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP P. dejongeii RVM04532 (37) VLAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP RSM05666_Sm (32) VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP RSM02162_Sm (39) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP At-Q8UAC0 (32) VLEAELAGKAKVHP--EGLGGRTTCYDDHAGPACRNGARALEVALSCHMP At-Q8UFG4 (33) VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M1-RMLO00301 (45) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP S261_M2aA12 (32) ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP M091_M4aE11 (33) ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADRNGARVLPTLLESHSP Consensus (51) VL A LGG   VIE  EGL GRTTAHDD  A   RNGAR LPT L SHAP 101                                            150 ACT MSMEG (84) LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLVUQVLUSAGGVGUUYTA Q98MY5 Mesorhizobium loti (79) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWPAP- Sma1993 Sinorhizobium (97) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA meliloti Q92XZ1 Sinorhizobium meliloti (87) LDLIIIMLGTNDLKRRFNMPPSEVA--MGIGCLVHDIRELSPGRTGN--- P. dejongeii RVM04532 (84) LDLVILMLGTNDLKSTFNVPPGEIA--AGAGVLGRMILAGDAGPENR-PP RSM05666_Sm (80) LDLIVFMLGSNDMKPIIHGTAFGAV--KGIERLVNLVRRHDWPTETEEG- RSM02162_Sm (86) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA At-Q8UAC0 (80) LDLVIIMLGTNDIKPVHGGRAEAAVS--GMRRLAQIVETFIYKPREAVP- At-Q8UFG4 (83) LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDWEAP M1-RMLO00301 (93) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWPAP- S261_M2aA12 (80) LDLVIIMLGTNDIKFAARCRAFDAS--MGMERLIQIVRSANYMKGYKIP- M091_M4aE11 (81) LDLIVIMLGTNDIKPHHGRTAGEAG--RGMARLVQIIRGHYAGRMQDEP- Consensus (101) LDLVIIMLGTNDMKP  H  P EAA   GM RLV IVR   YG     P 151                                            200 ACT MSMEG (132) PKVLVVSPPPLAPMPHPWFQLIFE--GGEQKUUELARVYSALASFMKVPF Q98MY5 Mesorhizobium loti (126) -QILIVSPPVVSRTENADFREMFAG--GDEASKQLAPQYAALADEVGCGF Sma1993 Sinorhizobium (145) PKVLWAPPPLAPMPDPWFEGMFG--GGYEKSKELSGLYKALADFMKVEF meliloti Q92XZ1 Sinorhizobium meliloti (132) DPEIMIVAPPPMLEDLKEWESIFS--GAQEKSRKLALEFEIMADSLEAHF P. dejongeii RVM04532 (131) QLLLMCPPKVRDLSAMPDLDAKIP--HGAARSAEFPRHYKAQAVALKCEY RSM05666_Sm (127) PEILIVSPPPLCETANSAFAAMFAG--GVEQSAMLAPLYRDLADELDCGF RSM02162_Sm (134) PKVLVVAPPPLAPMPDPWFEGMFG--GGYEKSKELSGLYKALADFMKVEF At-Q8UAC0 (127) -KLLIVAPPPCVAGPGGEPAGGRD----IEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (133) -DVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M1-RML000301 (140) -QILIVSPPVVSRTENADFREMFAG--GDEASKQLAPQYAALADEVGCGF S261_M2aA12 (127) -EILIISPPSLVPTQDEWFNDLWG--HAIAESKLFAKHYKRVAEELKVHF M091_M4aE11 (128) -QIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF Consensus (151)   ILIVSPPPL  T   DF AMFG   G E SK LA  YKALADELK  F 201                                   241 ACT MSMEG (180) FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL---- (SEQ ID NO: 662) Q98MY5 Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVMLEL-- (SEQ ID NO: 663) Sma1993 Sinorhizobium (193) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 664) meliloti Q92XZ1 Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO: 665) P. dejongeii RVM04532 (179) FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG--- (SEQ ID NO: 666) RSM05666_Sm (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPVVRMMLGL-- (SEQ ID NO: 667) RSM02162_Sm (182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 668) At-Q8UAC0 (172) FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG--- (SEQ ID NO: 669) At-Q8UFG4 (182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL-- (SEQ ID NO: 670) M1-RMLO00301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML---- (SEQ ID NO: 671) S261_M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL-- (SEQ ID NO: 672) M091_M4aE11 (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL-- (SEQ ID NO: 673) Consensus (201) FDAGTVA TSPVDGIHLDAENTR IG ALA VVR LLG (SEQ ID NO: 674)

A guide tree (i.e., an approximation of a phylogenetic tree) of the CLUSTALW alignment of active clonessequences is provided below.

TABLE 13-2 Similarity and Identity of Clones/Sequences Compared to M. smegmatis Perhydrolase % % Clone/Sequence Identity Similarity Sinorhizobium meliloti Sma1993 55.5 71.6 Sinorhizobium meliloti Q92XZ1 38.7 54.7 Mesorhizobium loti Q98MY5 38.8 53.4 Moraxella bovis AAK53448 5.0 9.7 Agrobacterium tumefaciens Q8UACO 36.7 47.7 Agrobacterium tumefaciens Q8UFG4 37.1 50.4 Mesorhizobium loti RMLO00301 34.8 50.9 Sinorhizobium meliloti RSM05666 37.4 52.5 Sinorhizobium meliloti RSM02162 58.3 75.2 Prosthecobacter dejongeii RVM05432 41.6 55.7 S261_M2aA12 39.3 54.3 M091_M4aE11 34.7 50.2

Based on the results, the active clones were found to have an overall identity to M. smegmatis perhydrolase of 38.7-58.3%. Moraxella bovis AAK53448 was found to be an exception and the (translated) amino acid sequence is questionable.

Redundancy

From the analyses above, it was evident that some redundancy exists in the alignment provided at the beginning of this Example that will have added undue weighting to the consensus sequence. Also, further GDSL-GRTT sequences were added. Thus, in the revised alignment below, the following changes were made:

Removed:

Natural isolate 14B

Natural isolate 2D

RSM02162_Sm

Q98MY5 Mesorhizobium loti

Added:

BAB16197 (Arh II)

BAB16192 (Arh I)

NP 00197751 (Mlo II)

NP 00216984 (Bce)

NP 522806 (Rso)

Non-redundant alignment:

1                                               50 20A (1) -------------LPSGILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG 9B Natural Isolate (1) -GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFP-KRVRWTG M. parafortuitum CO1 (1) -------------MAKRILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG MSAT (1) -------------MAKRILCFGDSLTWGWVPVEDGAPTERFA-PDVRWTG Sm-RSM05666 (1) --------------MKTVLCYGDSLTWGYDATG-----SGRHALEDRWPS At-Q8UAC0 (1) --------------MKTVLAFGDSLTWGADPAT-----GLRHPVEHRWPD At-Q8UFG4 (1) -------------MVKSVLCFGDSLTWGSNAET-----GGRHSHDDLWPS M091_M4aE11 (1) --------------MKTILAYGDSLTYGANPIP----GGPRHAYEDRWPT M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAE------GGRHALEDRWPS P. dejongeii RVM04532 (1) --------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhizobium meliloti (1) ---------MEETVARTVLCFGDSNTHGQVPG--RGPLDRYR-REQRWGG S261_M2aA12 (1) --------------MKNILAFGDSLTWGFVAG-----QDARHPFETRWPN Sma1993 Sinorhizobium meliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPTLRYP-YEQRWTG ZP_00197751 (1) --------------MKTILCYGDSLTWGYDAVG-----PSRHAYEDRWPS ZP_00216984 (1) ----------MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFA-REERWTG BAB16192 (1) -----MICHKGGEEMRSVLCYGDSNTHGQIPG--GSPLDRYG-PNERWPG BAB16197 (1) -----------MAESRSILCFGDSLTWGWIPVPESSPTLRYP-FEQRWTG NP_522806 (1) --------------MQQILLYSDSLSWGIIPG-----TRARLPFAARWAG Consensus (1)               MKTILCFGDSLTWGWIPV    P  RR   E RW G 51                                             100 20A (37) VLADLLGDRYEVIE---EGLSARTTTADDPADPRLN-GSQYLPSCLASHL 9B Natural Isolate (49) VLADELGAGYEVVE---EGLSARTTTADDPTDPRLN-GSDYLPACLASHL M. parafortuitum CO1 (37) VLADLLGDRYEVIE---EGLSARTTTAEDPADPRLN-GSQYLPSCLASHL MSAT (37) VLAQQLGADFEVIE---EGLSARTTNIDDPTDPRLN-GASYLPSCLATHL Sm-RSM05666 (32) VLQKALGSDAHVIA---EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHA At-Q8UAC0 (32) VLEAELAGKAKVHP---EGLGGRTTCYDDHAGPACANGARALEVALSCHM At-Q8UFG4 (33) VLQKALGSDVHVIFT-HEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHA M091_M4aE11 (33) ALEQGLGGKARVIA---EGLGGRTTVHDDWFANADRNGARVLPTLLESHS Ml-RML000301 (45) VLQASLGGGVQVIA---DGLNGRTTAFDDHLAGADANGARLLPTALTTHA P. dejongeii RVM04532 (37) VLAKALGAGFRVIE---EGQNGRTTVHEDPLNICRK-GKDYLPACLESHK Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE---EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHA S261_M2aA12 (32) ALAAGLGGKARVIE---EGQNGRTTVFDDAATFESRNGSVALPLLLISHQ Sma1993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE---EGLSARTTSLDDPNDARLN-GSTYLPMALASHL ZP_00197751 (32) VLQGRLGSSARVIA---EGLCGRTTAFDDWVAGADANGARILPTLLATHS ZP_00216984 (40) VLAQTLGASWRVIE---EGLPARTTVHDDPIEGRHKNGLSYLRACVESHL BAB16192 (43) VLRRELGSQWYVIE---EGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHA BAB16197 (39) AMAAALGDGYSIIE---EGLSARTTSVEDPNDPRLN-GSAYLPMALASHL NP_522806 (32) VMEHALQAQGHAVRIVEDCLNGRTTVLDDPARPGRN-GLQGLAQRIEAHA Consensus (51) VLA  LGA Y VIE   EGL GRIT  DDP D   RNGA YLP  L SH 101                                            150 20A (83) PLDLVILMLGINDTKANFGRTPFD--IATGMGVLATQVLTSAGG-VGTSY 9B Natural Isolate (95) PLDLVILMLGTNDTKANLNRTPVD--IASGMGVLATQVLTSAGG-VGTSY M. parafortuitum CO1 (83) PLDLVILMLGTNDTKANFGRTPFD--IATGMGVLATQVLTSAGG-VGTSY MSAT (83) PLDLVIIMLGTNDTKAYFRRTPLD--IALGMSVLVTQVLTSAGG-VGTTY Sm-RSM05666 (79) PLDLIVFMLGSNDMKPIIHGTAFG--AVKGIERLVNLVRRHDWPT--ETE At-Q8UAC0 (79) PLDLVIIMLGTNDIKPVHGGRAEA--AVSGMARLAQIVETFIYK---PRE At-Q8UFG4 (82) PLDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQV--SDW M091_M4aE11 (80) PLDLIVIMLGTNDIKPHHGRTAGE--AGRGMARLVQIIRGHYAG---RMQ Ml-RML000301 (92) PIDLIVIMLGANDMKPWIHGNPVA--AKQGIQRLIDIVRGHDYP---FDW P. dejongeii RVM04532 (83) PLDLVILMLGTNDLKSTFNVPPGE--IAAGAGVLGRMILAGDA---GPEN Q92XZ1 Sinorhizobium meliloti (86) PLDLIIIMLGTNDLKRRFNMPPSE--VAMGIGCLVHDIRELSP---GRTG S261_M2aA12 (79) PLDLVIIMLGTNDIKFAARCRAFD--ASMGMERLIQIVRSANYM---KGY Sma1993 Sinorhizobium meliloti (96) PLDLVIIMLGTNDTKSYFHRTPYE--IANGMGKLVGQVLTCAGG-VGTPY ZP_00197751 (79) PLDLVIVMLGTNDMKSFVCGRAIG--AKQGMERIVQIIRGQPYS---FNY ZP_00216984 (87) PVDVVVLMLGTNDLKTRFSVTPAD--IATSVGVLLAKIAACGA---GPSG BAB16192 (90) ILDLVIIMLGTNDLKARFGQPPSE--VAMGIGCLVYDIRELAP---GPGG BAB16197 (85) PLDLVIILLGTNDTKSYFRRTPYE--IANGMGKLAGQVLTSAGG-IGTPY NP_522806 (81) PLALVILMLGTNDFQAIFRHTAQD--AAQGVAQLVRAIRQAPIEP---GM Consensus (101) PLDLVIIMLGTNDLKA F  TP D  IA GMGRLV  VR   G   G  Y 151                                            200 20A (130) PAPQVLIVAPPPLGELPHPWFDL--VFSGGREKTAELARVYSALASFMKV 9B Natural Isolate (142) PAPQVLIVAPPPLAEMPHPWFEL--VFDGGREKTAQLARVYSALASFMKV M. parafortuitum CO1 (130) PAPQVLIVAPPPLGELPHPWFDL--VFSGGREKTAELARVYSALASFMKV MSAT (130) PAPKVLVVSPPPLAPMPHPWFQL--IFEGGEQKTTELARVYSALASFMKV Sm-RSM05666 (125) EGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAP--LYRDLADELDC At-Q8UAC0 (124) AVPKLLIVAPPPCVAGP--GGEPAGGRDIEQSMRLAP--LYRKLAAELGH At-Q8UFG4 (130) EAPDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDC M091_M4aE11 (125) DEPQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKV Ml-RML000301 (137) PAPQILIVSPPVVSRTENADFREMFAGGDEASKQLAP--QYAALADEVGC P. dejongeii RVM04532 (128) RPPQLLLMCPPKVRDLSAMPDLDAKIPHGAAR-SAEFPRHYKAQAVALKC Q92XZ1 Sinorhizobium meliloti (131) NDPEIMIVAPPPMLEDLKEWES---IFSGAQEKSRKLALEFEIMADSLEA S261_M2aA12 (124) KIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAK--HYKRVAEELKV Sma1993 Sinorhizobium meliloti (143) PAPKVLVVAPPPLAPMPDPWFEG--MFGGGYEKSKELSGLYKALADFMKV ZP_00197751 (124) KVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAP--LYAALAQQTGC ZP_00216984 (132) ASPKLVLMAPAPIVEVGFLGEI---FAGGAAK-SRQLAKRYEQVASDAGA BAB16192 (135) KPPEIMVVAPPPMLDDIKEWEP---IFSGAQEKSRRLALEFEIIADSLEV BAB16197 (132) PAPKLLIVSPPPLAPMPDPWFEG--MFGGGYEKSLELAKQYKALANFLKV NP_522806 (126) PVPPVLIVVPPAITAPAGAMADK---FADAQPKCAGLAQAYRATAQTLGC Consensus (151)  AP ILIVAPPPL E    WF    IFGGA  KS  LA  YKALA  LKV 201                                            248 20A (178) PFFDAGSVISTDGVDGTHFTRGETI----------------------- (SEQ ID NO: 675) 9B Natural Isolate (190) PFFDAGSVISTDGVDGTHFTRGETIDR--------------------- (SEQ ID NO: 676) M. parafortuitum CO1 (178) PFFDAGSVISTDGVDGIHFTRGEQST---------------------- (SEQ ID NO: 677) MSAT (178) PFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL--------- (SEQ ID NO: 678) Sm-RSM05666 (173) GFFDGGSVARTTPIDGVHLDAENTRAVGRGLEPVVRMMLGL------- (SEQ ID NO: 679) At-Q8UAC0 (170) HFFDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG-------- (SEQ ID NO: 680) At-Q8UFG4 (180) GFFDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL------- (SEQ ID NO: 681) M091_M4aE11 (175) HFFDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL------- (SEQ ID NO: 682) Ml-RML000301 (185) GFFDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML--------- (SEQ ID NO: 683) P. dejongeii RVM04532 (177) EYFFSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG-------- (SEQ ID NO: 684) Q92XZ1 Sinorhizobium meliloti (178) HFFDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA----- (SEQ ID NO: 685) S261_M2aA12 (172) HFFDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL------- (SEQ ID NO: 686) Sma1993 Sinorhizobium meliloti (191) EFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF--------- (SEQ ID NO: 687 ZP_00197751 (172) AFFDAGTVARTTPLDGIHLDAENTRAIGAGLEPVVRQALGL------- (SEQ ID NO: 688) ZP_00216984 (178) HFLDAGAIVEVSPVDGVHFAADQHRVLGQRVAALLQQIA--------- (SEQ ID NO: 689) BAB16192 (182) HFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGWR------- (SEQ ID NO: 690) BAB16197 (180) DFLDAGEFVKIDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAAA (SEQ ID NO: 691) NP_522806 (173) HVFDANSVTPASAVDGIHLDADQHAQLGRAMAQVVGTLLAQ------- (SEQ ID NO: 692) Consensus (201)  FFDAGSV  TSPVDGIHLDAENTR LG ALA  VR IL (SEQ ID NO: 693)

The guide tree to the CLUSTALW alignment (which approximates to a phylogenetic tree) clearly indicates 3 groupings:

1) GDSL-ARTT group including Act

2) GDSL-GRTT group composed of members of the Rhizobiales and the metagenome; and

3) Intermediate group of mixed motifs.

It is also contemplated that the results suggest some form of gene duplication and mutation events in the Rhizobiales and lateral gene transfer to Mycobacterium.

Using the non-redundant alignment a new Act consensus was constructed called “Act chimera”.

(SEQ ID NO: 694) 1 KTILCFGDSL TWGWIPVEDG APTERRAPEV RWTGVLAQQL GADYEVIEEG 51 LSGRTTNIDD PTDPRLRNGA SYLPSCLASH LPLDLVIIML GTNDLKAYFR 101 RTPLDIALGM GRLVTQVRTS AGGVGTTYPA PKILIVAPPP LAEMPHPWFQ 151 LIFGGAEQKS TELARVYKAL ASFLKVPFFD AGSVISTSPV DGIHLDAENT 201 RDLGVALAEQ VRSIL

An alignment of Act-chimera with Ms Act (Chimera align) indicates 91.6% similarity and 86.0% identity, as indicated below.

1                                               50 MSAT (1) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIE Act-Chimera (1) --KTILCFGDSLTWGWIPVEDGAPTERRAPEVRWTGVLAQQLGADYEVIE Consensus (1)   K ILCFGDSLTWGWIPVEDGAPTER APDVRWTGVLAQQLGADFEVIE 51                                             100 MSAT (51) EGLSARTTNIDDPTDPRLN-GASYLPSCLATHLPLDLVIIMLGTNDTKAY Act-Chimera (49) EGLSGRTTNIDDPTDPRLRNGASYLPSCLASHLPLDLVIIMLGTNDLKAY Consensus (51) EGLSARTTNIDDPTDPRL  GASYLPSCLASHLPLDLVIIMLGTND KAY 101                                            150 MSAT (100) FRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPW Act-Chimera (99) FRRTPLDIALGMGRLVTQVRTSAGGVGTTYPAPKILIVAPPPLAEMPHPW Consensus (101) FRRTPLDIALGM  LVTQV TSAGGVGTTYPAPKILIVAPPPLA MPHPW 151                                            200 MSAT (150) FQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEA Act-Chimera (149) FQLIFGGAEQKSTELARVYKALASFLKVPFFDAGSVISTSPVDGIHLDAE Consensus (151) FQLIF GAEQKSTELARVY ALASFLKVPFFDAGSVIST  VDGIH 201           217 MSAT (200) NNRDLGVALAEQVRSLL  (SEQ ID NO: 695) Act-Chimera (199) NTRDLGVALAEQVRSIL  (SEQ ID NO: 694) Consensus (201) N RDLGVALAEQVRSIL  (SEQ ID NO: 696)

A BLASTP search with Act-chimera did not reveal any further sequences.

The Act-chimera is “forced” on the Per sequence at the positions where no consensus exists. However, a basic ‘unforced’ consensus sequence did not provide any more information from a blastp search or from alignment analysis. Thus, comparison with the most distant homologues in the blastp ‘hit’ list was considered more useful in defining the important residuespositions in Act sequence space. This was a useful exercise, as these sequences were not used in the non-redundant alignment.

For example, Rhodopirellula baltica (NP_(—)865748; Psp; a Planctomycetes and quite different from either Mycobacterium or Rhizobiales), was compared as shown below.

1                                               50 MSAT (1) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLA---QQLGADFE NP_865746 (1) -MHSILIYGDSLSWGIIPGTR----RRFAFHQRWPGVMEIELRQTGIDAR Consensus (1)     IL FGDSLSWG IP      RFA   RW GVL     Q G D 51                                             100 MSAT (48) VIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTK NP_865746 (46) VIEDCLNGRRTVLEDPIKPGRNGLDGLQQRIEINSPLSLVVLFLGTNDFQ Consensus (51) VIED L AR T IDDP  P  NG   L   I    PL LVII LGTND 101                                            150 MSAT (98) AYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPH NP_865746 (96) SVHEFHAEQSAQGLALLV--DAIRRSPFEPGMPTPKILLVAPPTVHH-PK Consensus (101) A        A GLALLV              P PKILLVAPP L   P 151                                            200 MSAT (148) PWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFT NP_865746 (143) LDMAAKFQNAETKSTGLADAIRKVSTEHSCEFFDAATVTTTSVVDGVHLD Consensus (151)       F  AE KST LA     LAS     FFDAASV ST  VDGIH 201                222 MSAT (198) EANNRDLGVALAEQVRSLL---  (SEQ ID NO: 695) NP_865746 (193) QEQHQALGTALASTIAEILADC  (SEQ ID NO: 697) Consensus (201)   N   LG ALA  I  IL  (SEQ ID NO: 698) The following is an alignment with Ralstonia eutropha (Reu):

1                                               50 MSAT (1) ---------MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLA-- ZP_00166901 (1) MPLTAPSEVDPLQILVYADSLSWGIVPGTR----RRLPFPVRWPGRLELG Consensus (1)              IL FADSLSWG VP        R    VRW G L 51                                             100 MSAT (40) --QQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLV ZP_00166901 (47) LNADGGAPVRIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALV Consensus (51)      GA   IIED L AR T  DDP  P  NG   L   I  H PL LV 101                                            150 MSAT (88) IIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVV ZP_00166901 (97) VLMLGNNDFQSMHPHNAWHAAQGVGALV--HAIRTAPIEPGMPVPPILVV Consensus (101) IIMLG ND  A         A GM  LV       A I    P P ILVV 151                                            200 MSAT (138) SPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVIS ZP_00166901 (145) VPPPIRT-PCGPLAPKFAGGEHKWAGLPEALRELCATVDCSLFDAGTVIQ Consensus (151) PPPI   P       F GGE K   L      L A M    FDAGSVI 201                               237 MSAT (188) TDGVDGIHFTEANNRDLGVALAEQVRSLL-------- (SEQ ID NO: 695) ZP_00166901 (194) SSAVDGVHLDADAHVALGDALQPVVRALLAESSGHPS (SEQ ID NO: 699) Consensus (201) S AVDGIH        LG AL   VRALL (SEQ ID NO: 700)

Based on these results, the following conclusions were made. A BLASTp nr-database search with a perhydrolase consensus sequence revealed GDSL or GDSI lipasesesterases from a wide diversity of organisms. However, only 12 or 14 of these were reliable homologues of Per. Nearly all of these were derived from 1 small group of bacteria, namely the Rhizobiales (i.e., Gram-negative soil bacteria belonging the alpha-Proteobacteria). A few members of the beta-Proteobacteria were found, but no Mycobacterium sp. This provides an indication that the perhydrolase (Per) geneprotein is not widely distributed in nature.

The Mycobacterium protein is characterized by the GDSL-ARTT motif, whereas most of the Rhizobiales are characterized by a GDSL-GRTT motif. There are also some mixed or intermediate motifs (e.g., GDSN-GRTT, GDSN-ARTT and SDSL-GRTT). This may indicate gene duplication and mutation event and lateral gene transfer. The consensus residues identified in these experiments were L6, W14, R27, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, and G205.

Using the non-redundant alignment and comparison with distant homologues the follow sequence space can be defined starting at position 5 of the M. smegmatis perhydrolase and ending at position 195, with perhydrolase shown in residues in bold. [I, V][L][X][F,Y][G, S][D][S][L, N][T,S][W, Y, H][G][X]₂[P, A][X]₁₄[R, L][W][X]₇[L][X]₅[V, I][I, V, H][α][E. D][G, C][L,Q][X][G, A][R][T][T][X]₂[D, E][D][X]₇[G][X]₃[L][X]₆[H][X][P,I][L, I, V][D,A][V,I][X]₂[M,L][L][G][X][N][D][X]₃₆[P][X]₆[P][P, A][X]₃₁[A][X]₁₉[D][G][X][H] (SEQ ID NO:701)

In sum, it is clear from the analyses above that the active clonessequences with a GDSx₁-X₂RTT-Gx₃ND motif have all been found among the alpha-Proteobacteria-Gram-negative bacteria associated with the soil rhizosphere. This is in sharp contrast to the prototype perhydrolase from M. smegmatis a high GC content Gram-positive bacterium assigned to the class Actinobacteria. This division is illustrated in FIG. 2, which provides a phylogenetic tree, showing the major branches of the bacteria and the origin of the active clonessequences compared to M. smegmatis.

Example 14 Native Molecular Weight Estimation of Homologues of the Perhydrolase

In this Example, experiments conducted to estimate the native molecular weights of M. smegmatis perhydrolase homologues are described.

Preparation of Samples for Purification (Size Determination)

A single colony of the desired strains was inoculated in 50 ml Terrific Broth and incubated overnight at 37° C. with shaking at 200 rpm. The cells were pelleted by centrifugation for 10 minutes at 7000 rpm in a Sorvall SuperSpeed Centrifuge. The pellets were then resuspended in 10 ml 25 mM Bis-Tris (pH 6.5) and lysed by passage through a French pressure cell twice. The lysates were then centrifuged at 15000 rpm in a Sorvall SuperSpeed Centrifuge. The soluble fraction was heat treated at 55° C. for 1 hour to precipitate cellular proteins. The samples were then centrifuged at 10000 rpm in a Sorvall SuperSpeed Centrifuge and the soluble fractions used for further purification or assay.

Sizing Columns

The supernatants (prepared as described above) were run on a Sephadex 200 sizing column in 20 mM phosphate (pH 8.0), with a flow rate of 0.5 ml/min. The column was calibrated prior to running the samples with MW standards (listed below) and purified M. smegmatis perhydrolase protein. The crude sample elution volumes were determined by collecting 0.5 ml fractions, and assaying the fractions for pNB activity. Molecular weights and elution volumes of the standards:

Thyroglobulin MW 669 kDa: elution volume 16 ml Aldolase MW 158 kDa: elution volume 24 ml Ovalbumin MW 43 kDa: elution volume 26 ml Ribonuclease MW 14 kDa: elution volume 32 ml Perhydrolase elution volume 24 ml

Results

The following Table (Table 14-1) provides the elution volume of some of the M. smegmatis perhydrolase homologues identified herein.

TABLE 14-1 Elution Volume (Estimated Molecular Weight) of M. smegmatis Perhydrolase Homologues Homologue Sample Elution Volume (ml) pLO_SmeI 24 pET26_SmeII 24 pET26_MIO 24 pET26b_Stm 24 pET26b_Mbo 24 M7OaEB_pET26 32 pET26_m2aA12 24 pET26b_S2487am 32 S. meliloti RSM02162 (G00355) 24 PET_M2aAl2 (5261) 24 M. smegmatis Perhydrolase 24

The data in the above Table and the assay results obtained for these homologues indicated that these enzymes have an amino acid sequence similar to the M. smegmatis perhydrolase. As with the M. smegmatis perhydrolase, these homologues exhibit perhydrolysis activity as multimers. As described herein, the perhydrolase is an octamer, while the homologues, although they elute in a similar volume, are contemplated to be dimers, trimers, tetramers, hexamers, and or octamers.

Example 15 Crystal Structure of Perhydrolase

In this Example, the crystallographic analysis of the perhydrolase is described. Perhydrolase crystals were obtained under two conditions: 2.0 M [NH₄]₂SO₄, 2% PEG400, 0.1 M Tris pH 7.1 (giving triclinic, P1 crystals) and 1.0 M ammonium dihydrogen phosphate, and 0.1M sodium citrate pH 5.6 (giving tetragonal, P4 crystals) Both crystal forms gave suitable diffraction beyond 2.0 Å resolution. Derivative protein for a MAD phase determination using selenium replacing sulfur containing methionine resulting in a protein molecule having four selenomethionines the N-terminal methionine is cleaved proteolytically. Of the two forms, triclininc P1 a=83.77 Å b=90.07 Å c=112.115 Å α=73.32° β, 77.30° γ=88.07° and P4 a=b=98.18 Å c=230.12 Å, the P4 crystal gave data that was possible to use for structure determination. Three wavelength MAD datasets were collected at wavelengths corresponding to the Se absorption edge, near the inflection point and a third, away from the absorption edge.

Three hundred and thirty-three frames (0.3 degree oscillations per frame) for each wavelength with 1 sec exposure time were collected from a single tetragonal space group P4 crystal. The structure could be solved with either SOLVE or SHELX computer programs giving similar solutions for the 32 possible Se positions. The map was fitted using the program “O”. It was possible to trace electron density for residues 3-216 in each of the eight independent molecules. The final structure of these eight molecules was refined using CNS. The current crystallographic R-factor is 21%. The coordinates are provided below.

CRYST1 98.184 98.184 230.119 90.00 90.00 90.00 SCALE1 0.010185 0.000000 0.000000 0.000000 SCALE2 0.000000 0.010185 0.000000 0.000000 SCALE3 0.000000 0.000000 0.004346 0.000000 ATOM 1 CB LYS 3 −8.167 −61.964 18.588 1.000 40.95 ATOM 2 CG LYS 3 −8.685 −63.192 19.323 1.000 22.95 ATOM 3 CD LYS 3 −8.635 −64.400 18.399 1.000 14.97 ATOM 4 CE LYS 3 −7.963 −65.575 19.090 1.000 19.83 ATOM 5 NZ LYS 3 −7.359 −66.511 18.099 1.000 44.28 ATOM 6 C LYS 3 −9.684 −60.377 17.426 1.000 13.89 ATOM 7 O LYS 3 −9.087 −59.356 17.767 1.000 12.50 ATOM 8 N LYS 3 −8.000 −61.626 16.153 1.000 15.57 ATOM 9 CA LYS 3 −8.919 −61.686 17.284 1.000 20.71 ATOM 10 N ARG 4 −10.987 −60.381 17.166 1.000 24.56 ATOM 11 CA ARG 4 −11.695 −59.097 17.204 1.000 22.65 ATOM 12 CB ARG 4 −12.299 −58.822 15.822 1.000 21.44 ATOM 13 CG ARG 4 −11.232 −58.465 14.792 1.000 21.56 ATOM 14 CD ARG 4 −11.845 −58.181 13.431 1.000 29.29 ATOM 15 NE ARG 4 −11.660 −56.790 13.020 1.000 32.87 ATOM 16 CZ ARG 4 −12.643 −56.013 12.585 1.000 30.24 ATOM 17 NH1 ARG 4 −13.879 −56.487 12.494 1.000 17.82 ATOM 18 NH2 ARG 4 −12.399 −54.760 12.229 1.000 44.53 ATOM 19 C ARG 4 −12.735 −59.054 18.308 1.000 14.59 ATOM 20 O ARG 4 −13.604 −59.909 18.456 1.000 18.72 ATOM 21 N ILE 5 −12.639 −58.012 19.131 1.000 13.45 ATOM 22 CA ILE 5 −13.549 −57.882 20.263 1.000 12.08 ATOM 23 CB ILE 5 −12.747 −57.835 21.578 1.000 15.40 ATOM 24 CG2 ILE 5 −13.678 −57.677 22.765 1.000 5.80 ATOM 25 CG1 ILE 5 −11.811 −59.034 21.741 1.000 11.66 ATOM 26 CD1 ILE 5 −10.437 −58.632 22.232 1.000 19.35 ATOM 27 C ILE 5 −14.420 −56.640 20.142 1.000 8.96 ATOM 28 O ILE 5 −13.905 −55.529 20.021 1.000 13.31 ATOM 29 N LEU 6 −15.736 −56.833 20.169 1.000 13.04 ATOM 30 CA LEU 6 −16.675 −55.728 20.059 1.000 8.54 ATOM 31 CB LEU 6 −17.879 −56.087 19.178 1.000 7.42 ATOM 32 CG LEU 6 −18.959 −54.996 19.120 1.000 14.12 ATOM 33 CD1 LEU 6 −18.446 −53.783 18.359 1.000 12.19 ATOM 34 CD2 LEU 6 −20.245 −55.512 18.494 1.000 27.94 ATOM 35 C LEU 6 −17.170 −55.293 21.436 1.000 2.72 ATOM 36 O LEU 6 −17.719 −56.101 22.179 1.000 13.36 ATOM 37 N CYS 7 −16.978 −54.020 21.756 1.000 1.38 ATOM 38 CA CYS 7 −17.472 −53.469 23.011 1.000 3.17 ATOM 39 CB CYS 7 −16.411 −52.582 23.667 1.000 7.01 ATOM 40 SG CYS 7 −14.867 −53.471 23.992 1.000 11.21 ATOM 41 C CYS 7 −18.755 −52.685 22.776 1.000 0.65 ATOM 42 O CYS 7 −18.756 −51.627 22.145 1.000 4.76 ATOM 43 N PHE 8 −19.859 −53.228 23.281 1.000 0.00 ATOM 44 CA PHE 8 −21.147 −52.568 23.053 1.000 1.14 ATOM 45 CB PHE 8 −22.115 −53.578 22.443 1.000 5.54 ATOM 46 CG PHE 8 −23.421 −53.000 21.937 1.000 3.36 ATOM 47 CD1 PHE 8 −23.456 −52.212 20.800 1.000 0.89 ATOM 48 CD2 PHE 8 −24.602 −53.262 22.614 1.000 1.39 ATOM 49 CE1 PHE 8 −24.644 −51.683 20.333 1.000 0.00 ATOM 50 CE2 PHE 8 −25.793 −52.733 22.148 1.000 4.42 ATOM 51 CZ PHE 8 −25.818 −51.944 21.012 1.000 2.71 ATOM 52 C PHE 8 −21.677 −51.978 24.346 1.000 4.46 ATOM 53 O PHE 8 −21.873 −52.672 25.348 1.000 6.98 ATOM 54 N GLY 9 −21.923 −50.666 24.384 1.000 5.61 ATOM 55 CA GLY 9 −22.396 −50.109 25.646 1.000 5.44 ATOM 56 C GLY 9 −22.860 −48.673 25.522 1.000 5.66 ATOM 57 O GLY 9 −23.229 −48.222 24.440 1.000 14.54 ATOM 58 N ASP 10 −22.837 −47.964 26.641 1.000 3.89 ATOM 59 CA ASP 10 −23.322 −46.596 26.734 1.000 5.17 ATOM 60 CB ASP 10 −24.331 −46.467 27.880 1.000 2.99 ATOM 61 CG ASP 10 −23.807 −47.052 29.175 1.000 7.05 ATOM 62 OD1 ASP 10 −22.617 −46.829 29.494 1.000 17.93 ATOM 63 OD2 ASP 10 −24.564 −47.738 29.895 1.000 10.98 ATOM 64 C ASP 10 −22.154 −45.642 26.939 1.000 5.15 ATOM 65 O ASP 10 −21.022 −45.940 26.556 1.000 5.62 ATOM 66 N SER 11 −22.423 −44.497 27.554 1.000 9.02 ATOM 67 CA SER 11 −21.394 −43.493 27.802 1.000 3.43 ATOM 68 CB SER 11 −22.014 −42.331 28.585 1.000 7.25 ATOM 69 OG SER 11 −22.640 −42.813 29.763 1.000 18.93 ATOM 70 C SER 11 −20.199 −44.046 28.561 1.000 7.58 ATOM 71 O SER 11 −19.089 −43.508 28.501 1.000 16.71 ATOM 72 N LEU 12 −20.393 −45.133 29.308 1.000 6.56 ATOM 73 CA LEU 12 −19.264 −45.696 30.046 1.000 16.41 ATOM 74 CB LEU 12 −19.711 −46.759 31.042 1.000 17.05 ATOM 75 CG LEU 12 −20.598 −46.336 32.210 1.000 18.22 ATOM 76 CD1 LEU 12 −20.866 −47.527 33.123 1.000 7.48 ATOM 77 CD2 LEU 12 −19.973 −45.184 32.988 1.000 10.83 ATOM 78 C LEU 12 −18.269 −46.285 29.048 1.000 14.99 ATOM 79 O LEU 12 −17.065 −46.307 29.267 1.000 6.10 ATOM 80 N THR 13 −18.828 −46.764 27.940 1.000 14.77 ATOM 81 CA THR 13 −18.014 −47.347 26.876 1.000 8.83 ATOM 82 CB THR 13 −18.828 −48.381 26.080 1.000 6.87 ATOM 83 OG1 THR 13 −19.109 −49.487 26.949 1.000 10.08 ATOM 84 CG2 THR 13 −18.033 −48.940 24.914 1.000 16.85 ATOM 85 C THR 13 −17.490 −46.245 25.970 1.000 4.56 ATOM 86 O THR 13 −16.315 −46.220 25.616 1.000 11.71 ATOM 87 N TRP 14 −18.376 −45.317 25.612 1.000 5.57 ATOM 88 CA TRP 14 −17.992 −44.210 24.742 1.000 7.21 ATOM 89 CB TRP 14 −19.208 −43.329 24.453 1.000 6.90 ATOM 90 CG TRP 14 −18.917 −42.183 23.537 1.000 11.88 ATOM 91 CD2 TRP 14 −18.731 −40.813 23.924 1.000 13.72 ATOM 92 CE2 TRP 14 −18.483 −40.081 22.745 1.000 11.95 ATOM 93 CE3 TRP 14 −18.752 −40.147 25.152 1.000 10.63 ATOM 94 CD1 TRP 14 −18.779 −42.222 22.181 1.000 8.28 ATOM 95 NE1 TRP 14 −18.517 −40.963 21.694 1.000 7.16 ATOM 96 CZ2 TRP 14 −18.255 −38.705 22.763 1.000 5.39 ATOM 97 CZ3 TRP 14 −18.526 −38.783 25.168 1.000 12.55 ATOM 98 CH2 TRP 14 −18.282 −38.084 23.981 1.000 12.81 ATOM 99 C TRP 14 −16.880 −43.353 25.327 1.000 5.41 ATOM 100 O TRP 14 −16.107 −42.745 24.582 1.000 4.90 ATOM 101 N GLY 15 −16.794 −43.283 26.652 1.000 8.94 ATOM 102 CA GLY 15 −15.794 −42.475 27.318 1.000 4.51 ATOM 103 C GLY 15 −16.249 −41.098 27.755 1.000 10.98 ATOM 104 O GLY 15 −15.480 −40.136 27.646 1.000 15.11 ATOM 105 N TRP 16 −17.471 −40.952 28.255 1.000 23.34 ATOM 106 CA TRP 16 −17.988 −39.691 28.792 1.000 15.10 ATOM 107 CB TRP 16 −19.408 −39.890 29.327 1.000 6.11 ATOM 108 CG TRP 16 −20.139 −38.694 29.846 1.000 1.78 ATOM 109 CD2 TRP 16 −21.229 −38.008 29.213 1.000 8.98 ATOM 110 CE2 TRP 16 −21.613 −36.942 30.051 1.000 7.76 ATOM 111 CE3 TRP 16 −21.923 −38.186 28.009 1.000 15.66 ATOM 112 CD1 TRP 16 −19.927 −38.021 31.016 1.000 0.35 ATOM 113 NE1 TRP 16 −20.798 −36.973 31.154 1.000 8.35 ATOM 114 CZ2 TRP 16 −22.649 −36.063 29.734 1.000 5.16 ATOM 115 CZ3 TRP 16 −22.952 −37.317 27.692 1.000 5.34 ATOM 116 CH2 TRP 16 −23.306 −36.269 28.551 1.000 4.72 ATOM 117 C TRP 16 −17.059 −39.154 29.881 1.000 7.85 ATOM 118 O TRP 16 −16.846 −39.815 30.899 1.000 3.97 ATOM 119 N VAL 17 −16.533 −37.952 29.685 1.000 5.45 ATOM 120 CA VAL 17 −15.750 −37.256 30.695 1.000 12.08 ATOM 121 CB VAL 17 −14.822 −36.191 30.082 1.000 17.55 ATOM 122 CG1 VAL 17 −14.084 −35.443 31.185 1.000 11.59 ATOM 123 CG2 VAL 17 −13.841 −36.807 29.099 1.000 7.77 ATOM 124 C VAL 17 −16.673 −36.565 31.696 1.000 13.86 ATOM 125 O VAL 17 −17.390 −35.618 31.351 1.000 1.02 ATOM 126 N PRO 18 −16.660 −37.034 32.936 1.000 8.38 ATOM 127 CD PRO 18 −15.770 −38.071 33.476 1.000 8.64 ATOM 128 CA PRO 18 −17.572 −36.501 33.948 1.000 9.99 ATOM 129 CB PRO 18 −17.201 −37.294 35.208 1.000 12.31 ATOM 130 CG PRO 18 −15.817 −37.789 34.954 1.000 7.46 ATOM 131 C PRO 18 −17.327 −35.017 34.191 1.000 13.05 ATOM 132 O PRO 18 −16.163 −34.619 34.306 1.000 18.63 ATOM 133 N VAL 19 −18.381 −34.211 34.266 1.000 6.92 ATOM 134 CA VAL 19 −18.214 −32.793 34.585 1.000 9.29 ATOM 135 CB VAL 19 −18.482 −31.856 33.388 1.000 5.33 ATOM 136 CG1 VAL 19 −17.377 −31.995 32.354 1.000 6.78 ATOM 137 CG2 VAL 19 −19.850 −32.150 32.796 1.000 3.72 ATOM 138 C VAL 19 −19.151 −32.380 35.710 1.000 12.02 ATOM 139 O VAL 19 −20.217 −32.962 35.913 1.000 14.52 ATOM 140 N GLU 20 −18.771 −31.351 36.467 1.000 17.17 ATOM 141 CA GLU 20 −19.662 −30.994 37.575 1.000 13.30 ATOM 142 CB GLU 20 −18.918 −30.130 38.595 1.000 25.34 ATOM 143 CG GLU 20 −18.276 −30.968 39.702 1.000 31.46 ATOM 144 CD GLU 20 −16.871 −30.487 40.017 1.000 35.91 ATOM 145 OE1 GLU 20 −16.143 −30.157 39.055 1.000 40.11 ATOM 146 OE2 GLU 20 −16.507 −30.431 41.210 1.000 45.47 ATOM 147 C GLU 20 −20.913 −30.294 37.080 1.000 7.56 ATOM 148 O GLU 20 −21.964 −30.361 37.723 1.000 11.30 ATOM 149 N ASP 21 −20.852 −29.610 35.936 1.000 19.38 ATOM 150 CA ASP 21 −22.099 −28.983 35.471 1.000 23.47 ATOM 151 CB ASP 21 −21.815 −27.740 34.640 1.000 17.53 ATOM 152 CG ASP 21 −21.114 −27.991 33.326 1.000 14.93 ATOM 153 OD1 ASP 21 −20.984 −29.159 32.908 1.000 26.78 ATOM 154 OD2 ASP 21 −20.685 −26.996 32.694 1.000 8.74 ATOM 155 C ASP 21 −22.959 −29.988 34.707 1.000 19.54 ATOM 156 O ASP 21 −23.988 −29.627 34.131 1.000 22.49 ATOM 157 N GLY 22 −22.550 −31.250 34.697 1.000 13.19 ATOM 158 CA GLY 22 −23.279 −32.377 34.166 1.000 15.71 ATOM 159 C GLY 22 −23.507 −32.377 32.659 1.000 20.02 ATOM 160 O GLY 22 −23.370 −33.431 32.036 1.000 23.32 ATOM 161 N ALA 23 −23.846 −31.235 32.138 1.000 26.40 ATOM 162 CA ALA 23 −24.265 −30.672 30.873 1.000 28.79 ATOM 163 CB ALA 23 −24.483 −29.192 31.152 1.000 32.86 ATOM 164 C ALA 23 −23.309 −30.988 29.745 1.000 22.68 ATOM 165 O ALA 23 −22.922 −32.189 29.753 1.000 40.02 ATOM 166 N PRO 24 −22.847 −30.255 28.748 1.000 12.97 ATOM 167 CD PRO 24 −22.892 −28.855 28.309 1.000 15.92 ATOM 168 CA PRO 24 −22.051 −31.028 27.767 1.000 5.31 ATOM 169 CB PRO 24 −22.024 −30.134 26.520 1.000 4.03 ATOM 170 CG PRO 24 −22.002 −28.762 27.105 1.000 6.80 ATOM 171 C PRO 24 −20.622 −31.273 28.222 1.000 14.45 ATOM 172 O PRO 24 −20.034 −30.591 29.056 1.000 19.65 ATOM 173 N THR 25 −20.062 −32.310 27.600 1.000 13.21 ATOM 174 CA THR 25 −18.685 −32.690 27.894 1.000 11.82 ATOM 175 CB THR 25 −18.691 −33.772 28.987 1.000 12.19 ATOM 176 OG1 THR 25 −17.348 −34.104 29.355 1.000 19.38 ATOM 177 CG2 THR 25 −19.372 −35.027 28.454 1.000 0.00 ATOM 178 C THR 25 −18.009 −33.160 26.620 1.000 14.10 ATOM 179 O THR 25 −18.555 −33.019 25.518 1.000 16.46 ATOM 180 N GLU 26 −16.818 −33.724 26.762 1.000 12.30 ATOM 181 CA GLU 26 −16.157 −34.314 25.598 1.000 13.24 ATOM 182 CB GLU 26 −14.909 −33.518 25.225 1.000 15.75 ATOM 183 CG GLU 26 −15.211 −32.066 24.873 1.000 25.45 ATOM 184 CD GLU 26 −15.451 −31.152 26.056 1.000 27.41 ATOM 185 OE1 GLU 26 −14.687 −31.210 27.048 1.000 22.86 ATOM 186 OE2 GLU 26 −16.416 −30.347 26.012 1.000 17.32 ATOM 187 C GLU 26 −15.850 −35.775 25.891 1.000 8.80 ATOM 188 O GLU 26 −16.279 −36.316 26.909 1.000 2.55 ATOM 189 N ARG 27 −15.121 −36.421 25.001 1.000 13.28 ATOM 190 CA ARG 27 −14.783 −37.838 25.124 1.000 12.71 ATOM 191 CB ARG 27 −14.857 −38.447 23.726 1.000 6.07 ATOM 192 CG ARG 27 −14.491 −39.908 23.585 1.000 4.38 ATOM 193 CD ARG 27 −14.879 −40.387 22.186 1.000 11.29 ATOM 194 NE ARG 27 −14.974 −41.840 22.110 1.000 13.10 ATOM 195 CZ ARG 27 −15.191 −42.517 20.992 1.000 9.74 ATOM 196 NH1 ARG 27 −15.337 −41.868 19.842 1.000 11.38 ATOM 197 NH2 ARG 27 −15.262 −43.839 21.029 1.000 0.00 ATOM 198 C ARG 27 −13.413 −38.031 25.746 1.000 8.79 ATOM 199 O ARG 27 −12.534 −37.181 25.579 1.000 17.59 ATOM 200 N PHE 28 −13.183 −39.133 26.461 1.000 12.29 ATOM 201 CA PHE 28 −11.826 −39.379 26.955 1.000 9.91 ATOM 202 CB PHE 28 −11.783 −40.575 27.900 1.000 10.13 ATOM 203 CG PHE 28 −12.084 −40.263 29.355 1.000 11.54 ATOM 204 CD1 PHE 28 −11.250 −39.431 30.084 1.000 8.88 ATOM 205 CD2 PHE 28 −13.194 −40.802 29.979 1.000 11.27 ATOM 206 CE1 PHE 28 −11.535 −39.156 31.408 1.000 8.90 ATOM 207 CE2 PHE 28 −13.486 −40.533 31.305 1.000 5.41 ATOM 208 CZ PHE 28 −12.647 −39.703 32.020 1.000 0.61 ATOM 209 C PHE 28 −10.901 −39.635 25.770 1.000 11.56 ATOM 210 O PHE 28 −11.370 −40.112 24.736 1.000 13.14 ATOM 211 N ALA 29 −9.612 −39.349 25.896 1.000 13.02 ATOM 212 CA ALA 29 −8.674 −39.656 24.818 1.000 13.91 ATOM 213 CB ALA 29 −7.275 −39.163 25.151 1.000 6.49 ATOM 214 C ALA 29 −8.662 −41.157 24.545 1.000 15.68 ATOM 215 O ALA 29 −8.937 −41.954 25.446 1.000 31.74 ATOM 216 N PRO 30 −8.345 −41.537 23.314 1.000 11.44 ATOM 217 CD PRO 30 −7.982 −40.660 22.192 1.000 12.10 ATOM 218 CA PRO 30 −8.326 −42.955 22.936 1.000 18.85 ATOM 219 CB PRO 30 −7.822 −42.956 21.494 1.000 16.38 ATOM 220 CG PRO 30 −7.283 −41.593 21.244 1.000 14.74 ATOM 221 C PRO 30 −7.386 −43.767 23.826 1.000 13.40 ATOM 222 O PRO 30 −7.570 −44.969 23.979 1.000 8.18 ATOM 223 N ASP 31 −6.396 −43.115 24.412 1.000 22.50 ATOM 224 CA ASP 31 −5.426 −43.715 25.312 1.000 26.63 ATOM 225 CB ASP 31 −4.170 −42.841 25.398 1.000 30.41 ATOM 226 CG ASP 31 −3.792 −42.143 24.108 1.000 39.21 ATOM 227 OD1 ASP 31 −2.577 −42.086 23.802 1.000 39.00 ATOM 228 OD2 ASP 31 −4.673 −41.634 23.375 1.000 37.50 ATOM 229 C ASP 31 −5.985 −43.926 26.721 1.000 17.49 ATOM 230 O ASP 31 −5.482 −44.784 27.450 1.000 25.27 ATOM 231 N VAL 32 −6.989 −43.150 27.092 1.000 14.45 ATOM 232 CA VAL 32 −7.592 −43.125 28.421 1.000 12.64 ATOM 233 CB VAL 32 −7.966 −41.683 28.814 1.000 10.68 ATOM 234 CG1 VAL 32 −8.580 −41.609 30.199 1.000 13.66 ATOM 235 CG2 VAL 32 −6.742 −40.774 28.752 1.000 20.51 ATOM 236 C VAL 32 −8.808 −44.042 28.507 1.000 9.73 ATOM 237 O VAL 32 −8.890 −44.834 29.452 1.000 2.23 ATOM 238 N ARG 33 −9.722 −43.964 27.553 1.000 10.63 ATOM 239 CA ARG 33 −10.888 −44.824 27.410 1.000 6.85 ATOM 240 CB ARG 33 −11.369 −44.833 25.961 1.000 16.41 ATOM 241 CG ARG 33 −12.281 −43.727 25.488 1.000 21.19 ATOM 242 CD ARG 33 −12.464 −43.806 23.974 1.000 26.66 ATOM 243 NE ARG 33 −11.862 −42.659 23.309 1.000 30.35 ATOM 244 CZ ARG 33 −11.493 −42.567 22.044 1.000 31.60 ATOM 245 NH1 ARG 33 −11.658 −43.585 21.214 1.000 34.85 ATOM 246 NH2 ARG 33 −10.952 −41.433 21.610 1.000 52.70 ATOM 247 C ARG 33 −10.600 −46.279 27.775 1.000 9.71 ATOM 248 O ARG 33 −9.603 −46.830 27.300 1.000 16.85 ATOM 249 N TRP 34 −11.450 −46.924 28.577 1.000 10.64 ATOM 250 CA TRP 34 −11.166 −48.311 28.952 1.000 6.46 ATOM 251 CB TRP 34 −12.149 −48.855 29.979 1.000 12.45 ATOM 252 CG TRP 34 −13.561 −49.106 29.583 1.000 6.95 ATOM 253 CD2 TRP 34 −14.104 −50.199 28.835 1.000 9.27 ATOM 254 CE2 TRP 34 −15.493 −49.986 28.723 1.000 5.43 ATOM 255 CE3 TRP 34 −13.571 −51.345 28.240 1.000 14.72 ATOM 256 CD1 TRP 34 −14.622 −48.298 29.888 1.000 4.49 ATOM 257 NE1 TRP 34 −15.786 −48.820 29.374 1.000 4.03 ATOM 258 CZ2 TRP 34 −16.337 −50.864 28.050 1.000 8.19 ATOM 259 CZ3 TRP 34 −14.405 −52.216 27.572 1.000 12.73 ATOM 260 CH2 TRP 34 −15.778 −51.976 27.479 1.000 8.32 ATOM 261 C TRP 34 −11.111 −49.214 27.723 1.000 7.27 ATOM 262 O TRP 34 −10.393 −50.222 27.767 1.000 11.53 ATOM 263 N THR 35 −11.839 −48.887 26.659 1.000 1.15 ATOM 264 CA THR 35 −11.730 −49.673 25.431 1.000 5.29 ATOM 265 CB THR 35 −12.708 −49.239 24.331 1.000 3.10 ATOM 266 OG1 THR 35 −12.629 −47.820 24.163 1.000 15.85 ATOM 267 CG2 THR 35 −14.146 −49.549 24.726 1.000 5.16 ATOM 268 C THR 35 −10.307 −49.555 24.882 1.000 14.32 ATOM 269 O THR 35 −9.738 −50.494 24.333 1.000 12.77 ATOM 270 N GLY 36 −9.756 −48.361 25.060 1.000 15.72 ATOM 271 CA GLY 36 −8.392 −48.056 24.689 1.000 15.87 ATOM 272 C GLY 36 −7.407 −48.785 25.583 1.000 14.86 ATOM 273 O GLY 36 −6.374 −49.252 25.101 1.000 22.97 ATOM 274 N VAL 37 −7.686 −48.905 26.884 1.000 12.48 ATOM 275 CA VAL 37 −6.696 −49.577 27.728 1.000 11.76 ATOM 276 CB VAL 37 −6.921 −49.365 29.229 1.000 10.95 ATOM 277 CG1 VAL 37 −6.092 −50.382 30.009 1.000 0.00 ATOM 278 CG2 VAL 37 −6.577 −47.940 29.630 1.000 10.31 ATOM 279 C VAL 37 −6.707 −51.081 27.471 1.000 16.75 ATOM 280 O VAL 37 −5.669 −51.735 27.494 1.000 14.29 ATOM 281 N LEU 38 −7.911 −51.586 27.238 1.000 14.60 ATOM 282 CA LEU 38 −8.094 −52.999 26.917 1.000 11.25 ATOM 283 CB LEU 38 −9.573 −53.266 26.660 1.000 12.92 ATOM 284 CG LEU 38 −9.975 −54.663 26.198 1.000 15.77 ATOM 285 CD1 LEU 38 −9.747 −55.691 27.293 1.000 0.00 ATOM 286 CD2 LEU 38 −11.425 −54.677 25.733 1.000 24.28 ATOM 287 C LEU 38 −7.224 −53.347 25.720 1.000 7.67 ATOM 288 O LEU 38 −6.408 −54.262 25.740 1.000 13.04 ATOM 289 N ALA 39 −7.404 −52.568 24.659 1.000 9.64 ATOM 290 CA ALA 39 −6.603 −52.667 23.451 1.000 3.53 ATOM 291 CB ALA 39 −6.894 −51.487 22.530 1.000 6.32 ATOM 292 C ALA 39 −5.112 −52.704 23.761 1.000 9.32 ATOM 293 O ALA 39 −4.411 −53.632 23.367 1.000 28.59 ATOM 294 N GLN 40 −4.653 −51.665 24.456 1.000 21.51 ATOM 295 CA GLN 40 −3.251 −51.553 24.833 1.000 18.93 ATOM 296 CB GLN 40 −2.974 −50.365 25.744 1.000 28.00 ATOM 297 CG GLN 40 −3.597 −49.034 25.378 1.000 37.51 ATOM 298 CD GLN 40 −3.070 −47.877 26.214 1.000 40.85 ATOM 299 OE1 GLN 40 −1.998 −47.335 25.933 1.000 61.34 ATOM 300 NE2 GLN 40 −3.809 −47.475 27.248 1.000 9.83 ATOM 301 C GLN 40 −2.822 −52.851 25.525 1.000 10.96 ATOM 302 O GLN 40 −1.856 −53.475 25.106 1.000 18.66 ATOM 303 N GLN 41 −3.563 −53.239 26.552 1.000 15.02 ATOM 304 CA GLN 41 −3.253 −54.423 27.337 1.000 22.27 ATOM 305 CB GLN 41 −4.258 −54.582 28.484 1.000 16.69 ATOM 306 CG GLN 41 −4.064 −53.605 29.624 1.000 14.55 ATOM 307 CD GLN 41 −2.788 −53.852 30.406 1.000 16.86 ATOM 308 OE1 GLN 41 −2.759 −54.650 31.344 1.000 13.75 ATOM 309 NE2 GLN 41 −1.731 −53.158 30.008 1.000 21.79 ATOM 310 C GLN 41 −3.261 −55.694 26.493 1.000 28.40 ATOM 311 O GLN 41 −2.442 −56.589 26.703 1.000 26.71 ATOM 312 N LEU 42 −4.190 −55.776 25.546 1.000 28.62 ATOM 313 CA LEU 42 −4.373 −57.007 24.780 1.000 26.50 ATOM 314 CB LEU 42 −5.707 −56.920 24.012 1.000 19.31 ATOM 315 CG LEU 42 −6.934 −57.122 24.914 1.000 16.32 ATOM 316 CD1 LEU 42 −8.226 −57.077 24.119 1.000 10.94 ATOM 317 CD2 LEU 42 −6.810 −58.438 25.673 1.000 15.03 ATOM 318 C LEU 42 −3.217 −57.312 23.846 1.000 23.29 ATOM 319 O LEU 42 −2.770 −58.457 23.728 1.000 20.82 ATOM 320 N GLY 43 −2.693 −56.312 23.141 1.000 22.18 ATOM 321 CA GLY 43 −1.605 −56.590 22.215 1.000 18.95 ATOM 322 C GLY 43 −2.086 −56.793 20.791 1.000 23.97 ATOM 323 O GLY 43 −3.284 −56.838 20.514 1.000 27.50 ATOM 324 N ALA 44 −1.136 −56.927 19.879 1.000 22.72 ATOM 325 CA ALA 44 −1.317 −57.012 18.448 1.000 24.25 ATOM 326 CB ALA 44 0.048 −56.939 17.755 1.000 13.44 ATOM 327 C ALA 44 −2.034 −58.272 17.990 1.000 23.83 ATOM 328 O ALA 44 −2.146 −58.520 16.787 1.000 17.77 ATOM 329 N ASP 45 −2.524 −59.086 18.917 1.000 21.59 ATOM 330 CA ASP 45 −3.230 −60.298 18.495 1.000 17.80 ATOM 331 CB ASP 45 −2.705 −61.491 19.296 1.000 18.22 ATOM 332 CG ASP 45 −1.201 −61.625 19.113 1.000 24.69 ATOM 333 OD1 ASP 45 −0.710 −61.174 18.053 1.000 34.10 ATOM 334 OD2 ASP 45 −0.517 −62.159 20.007 1.000 33.14 ATOM 335 C ASP 45 −4.732 −60.107 18.647 1.000 11.82 ATOM 336 O ASP 45 −5.535 −60.992 18.364 1.000 23.89 ATOM 337 N PHE 46 −5.097 −58.914 19.097 1.000 9.27 ATOM 338 CA PHE 46 −6.485 −58.519 19.253 1.000 12.25 ATOM 339 CB PHE 46 −6.909 −58.479 20.722 1.000 14.52 ATOM 340 CG PHE 46 −6.474 −59.693 21.529 1.000 11.99 ATOM 341 CD1 PHE 46 −5.160 −59.814 21.956 1.000 12.17 ATOM 342 CD2 PHE 46 −7.383 −60.690 21.846 1.000 8.34 ATOM 343 CE1 PHE 46 −4.760 −60.917 22.683 1.000 13.46 ATOM 344 CE2 PHE 46 −6.990 −61.794 22.575 1.000 6.30 ATOM 345 CZ PHE 46 −5.680 −61.904 22.998 1.000 8.44 ATOM 346 C PHE 46 −6.725 −57.149 18.615 1.000 13.30 ATOM 347 O PHE 46 −5.816 −56.366 18.366 1.000 27.22 ATOM 348 N GLU 47 −7.992 −56.883 18.349 1.000 12.78 ATOM 349 CA GLU 47 −8.469 −55.616 17.833 1.000 9.15 ATOM 350 CB GLU 47 −8.667 −55.644 16.325 1.000 11.20 ATOM 351 CG GLU 47 −8.791 −54.276 15.670 1.000 21.84 ATOM 352 CD GLU 47 −9.726 −54.293 14.474 1.000 25.88 ATOM 353 OE1 GLU 47 −9.575 −55.205 13.632 1.000 30.74 ATOM 354 OE2 GLU 47 −10.602 −53.408 14.388 1.000 7.59 ATOM 355 C GLU 47 −9.781 −55.280 18.550 1.000 11.37 ATOM 356 O GLU 47 −10.722 −56.071 18.545 1.000 11.73 ATOM 357 N VAL 48 −9.775 −54.103 19.160 1.000 10.53 ATOM 358 CA VAL 48 −10.954 −53.604 19.843 1.000 8.11 ATOM 359 CB VAL 48 −10.595 −52.826 21.115 1.000 9.71 ATOM 360 CG1 VAL 48 −11.842 −52.251 21.773 1.000 15.31 ATOM 361 CG2 VAL 48 −9.849 −53.732 22.085 1.000 7.41 ATOM 362 C VAL 48 −11.745 −52.714 18.882 1.000 12.72 ATOM 363 O VAL 48 −11.147 −51.879 18.203 1.000 10.16 ATOM 364 N ILE 49 −13.046 −52.943 18.862 1.000 13.04 ATOM 365 CA ILE 49 −14.031 −52.170 18.122 1.000 14.10 ATOM 366 CB ILE 49 −14.879 −53.068 17.203 1.000 16.77 ATOM 367 CG2 ILE 49 −15.735 −52.214 16.285 1.000 1.57 ATOM 368 CG1 ILE 49 −14.049 −54.081 16.415 1.000 18.10 ATOM 369 CD1 ILE 49 −14.687 −54.559 15.133 1.000 14.33 ATOM 370 C ILE 49 −14.930 −51.406 19.091 1.000 9.02 ATOM 371 O ILE 49 −15.531 −52.013 19.983 1.000 15.82 ATOM 372 N GLU 50 −15.000 −50.085 18.932 1.000 5.34 ATOM 373 CA GLU 50 −15.730 −49.277 19.911 1.000 12.03 ATOM 374 CB GLU 50 −14.967 −47.984 20.222 1.000 10.36 ATOM 375 CG GLU 50 −13.623 −48.203 20.889 1.000 7.32 ATOM 376 CD GLU 50 −12.768 −46.966 21.056 1.000 7.06 ATOM 377 OE1 GLU 50 −12.744 −46.077 20.177 1.000 5.78 ATOM 378 OE2 GLU 50 −12.079 −46.870 22.101 1.000 25.19 ATOM 379 C GLU 50 −17.145 −48.962 19.446 1.000 6.79 ATOM 380 O GLU 50 −17.358 −48.318 18.423 1.000 8.80 ATOM 381 N GLU 51 −18.118 −49.429 20.225 1.000 9.34 ATOM 382 CA GLU 51 −19.524 −49.179 19.924 1.000 16.23 ATOM 383 CB GLU 51 −20.173 −50.400 19.270 1.000 15.22 ATOM 384 CG GLU 51 −19.757 −50.596 17.820 1.000 18.39 ATOM 385 CD GLU 51 −20.348 −49.531 16.917 1.000 17.99 ATOM 386 OE1 GLU 51 −21.352 −48.912 17.332 1.000 26.29 ATOM 387 OE2 GLU 51 −19.820 −49.309 15.809 1.000 15.93 ATOM 388 C GLU 51 −20.295 −48.788 21.184 1.000 10.51 ATOM 389 O GLU 51 −21.202 −49.495 21.623 1.000 7.29 ATOM 390 N GLY 52 −19.906 −47.655 21.751 1.000 5.90 ATOM 391 CA GLY 52 −20.533 −47.140 22.961 1.000 3.93 ATOM 392 C GLY 52 −21.329 −45.887 22.635 1.000 6.21 ATOM 393 O GLY 52 −20.785 −44.950 22.057 1.000 16.40 ATOM 394 N LEU 53 −22.607 −45.890 22.989 1.000 11.68 ATOM 395 CA LEU 53 −23.498 −44.764 22.710 1.000 7.60 ATOM 396 CB LEU 53 −24.627 −45.195 21.792 1.000 4.45 ATOM 397 CG LEU 53 −25.576 −44.164 21.185 1.000 3.84 ATOM 398 CD1 LEU 53 −26.721 −43.872 22.141 1.000 15.09 ATOM 399 CD2 LEU 53 −24.856 −42.874 20.817 1.000 3.41 ATOM 400 C LEU 53 −24.035 −44.204 24.023 1.000 5.05 ATOM 401 O LEU 53 −24.664 −44.920 24.801 1.000 5.74 ATOM 402 N SER 54 −23.771 −42.918 24.251 1.000 9.85 ATOM 403 CA SER 54 −24.192 −42.296 25.502 1.000 10.24 ATOM 404 CB SER 54 −23.797 −40.819 25.524 1.000 7.63 ATOM 405 OG SER 54 −22.395 −40.683 25.640 1.000 4.65 ATOM 406 C SER 54 −25.695 −42.448 25.691 1.000 7.74 ATOM 407 O SER 54 −26.438 −42.326 24.717 1.000 10.39 ATOM 408 N ALA 55 −26.127 −42.713 26.920 1.000 0.00 ATOM 409 CA ALA 55 −27.554 −42.749 27.218 1.000 0.00 ATOM 410 CB ALA 55 −28.209 −41.474 26.713 1.000 0.00 ATOM 411 C ALA 55 −28.235 −43.982 26.640 1.000 6.11 ATOM 412 O ALA 55 −29.442 −44.179 26.816 1.000 2.57 ATOM 413 N ARG 56 −27.474 −44.843 25.971 1.000 8.50 ATOM 414 CA ARG 56 −27.997 −46.084 25.433 1.000 5.94 ATOM 415 CB ARG 56 −26.919 −46.868 24.672 1.000 0.00 ATOM 416 CG ARG 56 −27.420 −48.244 24.247 1.000 2.73 ATOM 417 CD ARG 56 −26.467 −48.951 23.307 1.000 0.00 ATOM 418 NE ARG 56 −26.552 −48.440 21.935 1.000 6.44 ATOM 419 CZ ARG 56 −25.465 −48.325 21.170 1.000 11.18 ATOM 420 NH1 ARG 56 −24.283 −48.678 21.666 1.000 0.00 ATOM 421 NH2 ARG 56 −25.549 −47.861 19.928 1.000 1.13 ATOM 422 C ARG 56 −28.539 −47.009 26.526 1.000 12.43 ATOM 423 O ARG 56 −27.886 −47.179 27.556 1.000 10.16 ATOM 424 N THR 57 −29.697 −47.592 26.262 1.000 9.24 ATOM 425 CA THR 57 −30.376 −48.548 27.120 1.000 9.36 ATOM 426 CB THR 57 −31.855 −48.161 27.315 1.000 4.78 ATOM 427 OG1 THR 57 −32.608 −48.509 26.146 1.000 3.70 ATOM 428 CG2 THR 57 −31.992 −46.656 27.484 1.000 0.00 ATOM 429 C THR 57 −30.284 −49.953 26.532 1.000 10.18 ATOM 430 O THR 57 −29.873 −50.099 25.378 1.000 12.60 ATOM 431 N THR 58 −30.648 −50.987 27.286 1.000 5.87 ATOM 432 CA THR 58 −30.574 −52.349 26.769 1.000 1.65 ATOM 433 CB THR 58 −30.850 −53.410 27.853 1.000 5.35 ATOM 434 OG1 THR 58 −32.151 −53.196 28.413 1.000 12.48 ATOM 435 CG2 THR 58 −29.859 −53.311 29.002 1.000 11.47 ATOM 436 C THR 58 −31.556 −52.569 25.624 1.000 1.31 ATOM 437 O THR 58 −31.162 −52.902 24.506 1.000 7.78 ATOM 438 N ASN 59 −32.856 −52.404 25.867 1.000 4.91 ATOM 439 CA ASN 59 −33.810 −52.604 24.772 1.000 11.25 ATOM 440 CB ASN 59 −34.150 −54.090 24.624 1.000 9.19 ATOM 441 CG ASN 59 −35.186 −54.548 25.629 1.000 9.50 ATOM 442 OD1 ASN 59 −35.293 −54.000 26.725 1.000 13.36 ATOM 443 ND2 ASN 59 −35.965 −55.556 25.263 1.000 4.31 ATOM 444 C ASN 59 −35.070 −51.775 24.960 1.000 8.67 ATOM 445 O ASN 59 −36.172 −52.160 24.574 1.000 12.75 ATOM 446 N ILE 60 −34.938 −50.587 25.548 1.000 10.46 ATOM 447 CA ILE 60 −36.128 −49.752 25.722 1.000 10.70 ATOM 448 CB ILE 60 −36.572 −49.721 27.198 1.000 11.36 ATOM 449 CG2 ILE 60 −35.465 −49.223 28.112 1.000 0.00 ATOM 450 CG1 ILE 60 −37.872 −48.940 27.417 1.000 8.05 ATOM 451 CD1 ILE 60 −38.291 −48.800 28.860 1.000 27.90 ATOM 452 C ILE 60 −35.879 −48.350 25.177 1.000 16.37 ATOM 453 O ILE 60 −34.813 −47.773 25.374 1.000 28.53 ATOM 454 N ASP 61 −36.861 −47.811 24.470 1.000 18.37 ATOM 455 CA ASP 61 −36.838 −46.520 23.821 1.000 12.62 ATOM 456 CB ASP 61 −38.110 −46.353 22.977 1.000 12.58 ATOM 457 CG ASP 61 −38.111 −47.199 21.725 1.000 12.09 ATOM 458 OD1 ASP 61 −37.044 −47.723 21.349 1.000 16.37 ATOM 459 OD2 ASP 61 −39.197 −47.332 21.122 1.000 23.20 ATOM 460 C ASP 61 −36.796 −45.350 24.794 1.000 11.54 ATOM 461 O ASP 61 −37.626 −45.279 25.702 1.000 8.66 ATOM 462 N ASP 62 −35.860 −44.428 24.603 1.000 8.03 ATOM 463 CA ASP 62 −35.844 −43.228 25.431 1.000 14.39 ATOM 464 CB ASP 62 −34.430 −42.656 25.565 1.000 13.94 ATOM 465 CG ASP 62 −34.384 −41.598 26.656 1.000 18.06 ATOM 466 OD1 ASP 62 −33.609 −41.768 27.622 1.000 13.05 ATOM 467 OD2 ASP 62 −35.129 −40.604 26.536 1.000 20.19 ATOM 468 C ASP 62 −36.759 −42.162 24.844 1.000 13.14 ATOM 469 O ASP 62 −36.506 −41.698 23.731 1.000 14.36 ATOM 470 N PRO 63 −37.800 −41.751 25.553 1.000 8.49 ATOM 471 CD PRO 63 −38.102 −42.088 26.951 1.000 4.73 ATOM 472 CA PRO 63 −38.805 −40.853 24.972 1.000 16.60 ATOM 473 CB PRO 63 −39.802 −40.646 26.123 1.000 11.61 ATOM 474 CG PRO 63 −39.020 −40.960 27.352 1.000 8.04 ATOM 475 C PRO 63 −38.251 −39.504 24.531 1.000 19.70 ATOM 476 O PRO 63 −38.924 −38.738 23.835 1.000 10.26 ATOM 477 N THR 64 −37.024 −39.180 24.922 1.000 22.29 ATOM 478 CA THR 64 −36.429 −37.908 24.534 1.000 19.30 ATOM 479 CB THR 64 −35.852 −37.191 25.769 1.000 20.62 ATOM 480 OG1 THR 64 −34.550 −37.713 26.045 1.000 30.42 ATOM 481 CG2 THR 64 −36.718 −37.467 26.992 1.000 7.89 ATOM 482 C THR 64 −35.329 −38.087 23.497 1.000 19.22 ATOM 483 O THR 64 −34.609 −37.132 23.183 1.000 11.15 ATOM 484 N ASP 65 −35.189 −39.301 22.965 1.000 15.61 ATOM 485 CA ASP 65 −34.139 −39.542 21.967 1.000 18.78 ATOM 486 CB ASP 65 −32.777 −39.286 22.605 1.000 20.50 ATOM 487 CG ASP 65 −31.613 −39.348 21.638 1.000 17.33 ATOM 488 OD1 ASP 65 −31.767 −39.935 20.550 1.000 19.33 ATOM 489 OD2 ASP 65 −30.538 −38.810 21.983 1.000 15.26 ATOM 490 C ASP 65 −34.241 −40.945 21.382 1.000 14.84 ATOM 491 O ASP 65 −33.982 −41.936 22.060 1.000 8.38 ATOM 492 N PRO 66 −34.638 −41.026 20.115 1.000 15.75 ATOM 493 CD PRO 66 −34.896 −39.870 19.235 1.000 23.61 ATOM 494 CA PRO 66 −34.882 −42.301 19.441 1.000 9.14 ATOM 495 CB PRO 66 −35.693 −41.871 18.206 1.000 14.38 ATOM 496 CG PRO 66 −35.210 −40.494 17.902 1.000 16.45 ATOM 497 C PRO 66 −33.621 −43.029 18.995 1.000 8.15 ATOM 498 O PRO 66 −33.695 −44.041 18.283 1.000 12.38 ATOM 499 N ARG 67 −32.446 −42.557 19.404 1.000 11.98 ATOM 500 CA ARG 67 −31.209 −43.225 19.020 1.000 7.77 ATOM 501 CB ARG 67 −30.081 −42.211 18.831 1.000 8.16 ATOM 502 CG ARG 67 −30.162 −41.308 17.614 1.000 7.27 ATOM 503 CD ARG 67 −29.078 −40.228 17.713 1.000 11.05 ATOM 504 NE ARG 67 −29.378 −39.266 18.769 1.000 11.17 ATOM 505 CZ ARG 67 −28.768 −38.115 19.001 1.000 13.35 ATOM 506 NH1 ARG 67 −27.756 −37.708 18.245 1.000 3.80 ATOM 507 NH2 ARG 67 −29.168 −37.347 20.010 1.000 9.93 ATOM 508 C ARG 67 −30.728 −44.239 20.048 1.000 8.92 ATOM 509 O ARG 67 −29.714 −44.887 19.774 1.000 13.65 ATOM 510 N LEU 68 −31.389 −44.365 21.191 1.000 9.14 ATOM 511 CA LEU 68 −30.805 −45.057 22.335 1.000 13.92 ATOM 512 CB LEU 68 −31.052 −44.223 23.608 1.000 7.80 ATOM 513 CG LEU 68 −30.899 −42.707 23.481 1.000 8.78 ATOM 514 CD1 LEU 68 −31.285 −41.987 24.770 1.000 13.12 ATOM 515 CD2 LEU 68 −29.477 −42.333 23.090 1.000 3.77 ATOM 516 C LEU 68 −31.299 −46.478 22.571 1.000 16.19 ATOM 517 O LEU 68 −30.895 −47.092 23.574 1.000 5.21 ATOM 518 N ASN 69 −32.139 −47.056 21.716 1.000 7.75 ATOM 519 CA ASN 69 −32.520 −48.457 21.927 1.000 6.53 ATOM 520 CB ASN 69 −33.807 −48.842 21.198 1.000 6.25 ATOM 521 CG ASN 69 −34.377 −50.172 21.658 1.000 11.70 ATOM 522 OD1 ASN 69 −33.732 −51.219 21.664 1.000 2.64 ATOM 523 ND2 ASN 69 −35.646 −50.164 22.057 1.000 10.84 ATOM 524 C ASN 69 −31.406 −49.404 21.480 1.000 8.62 ATOM 525 O ASN 69 −31.204 −49.617 20.287 1.000 14.61 ATOM 526 N GLY 70 −30.697 −49.972 22.452 1.000 8.79 ATOM 527 CA GLY 70 −29.582 −50.854 22.212 1.000 1.64 ATOM 528 C GLY 70 −29.911 −52.031 21.316 1.000 6.17 ATOM 529 O GLY 70 −29.189 −52.293 20.355 1.000 12.06 ATOM 530 N ALA 71 −30.982 −52.744 21.622 1.000 1.39 ATOM 531 CA ALA 71 −31.442 −53.885 20.843 1.000 5.92 ATOM 532 CB ALA 71 −32.688 −54.457 21.529 1.000 3.81 ATOM 533 C ALA 71 −31.766 −53.565 19.392 1.000 4.67 ATOM 534 O ALA 71 −31.565 −54.391 18.490 1.000 0.00 ATOM 535 N SER 72 −32.295 −52.371 19.121 1.000 3.88 ATOM 536 CA SER 72 −32.687 −52.033 17.752 1.000 6.33 ATOM 537 CB SER 72 −33.678 −50.870 17.759 1.000 4.05 ATOM 538 OG SER 72 −33.023 −49.637 18.004 1.000 25.62 ATOM 539 C SER 72 −31.468 −51.730 16.884 1.000 7.90 ATOM 540 O SER 72 −31.568 −51.720 15.658 1.000 12.06 ATOM 541 N TYR 73 −30.315 −51.505 17.498 1.000 8.51 ATOM 542 CA TYR 73 −29.070 −51.210 16.789 1.000 8.77 ATOM 543 CB TYR 73 −28.394 −50.029 17.478 1.000 10.31 ATOM 544 CG TYR 73 −27.124 −49.453 16.913 1.000 11.92 ATOM 545 CD1 TYR 73 −27.113 −48.329 16.090 1.000 8.49 ATOM 546 CE1 TYR 73 −25.931 −47.812 15.586 1.000 1.47 ATOM 547 CD2 TYR 73 −25.888 −50.018 17.201 1.000 10.36 ATOM 548 CE2 TYR 73 −24.704 −49.512 16.703 1.000 9.07 ATOM 549 CZ TYR 73 −24.727 −48.398 15.890 1.000 5.36 ATOM 550 OH TYR 73 −23.544 −47.902 15.391 1.000 10.80 ATOM 551 C TYR 73 −28.148 −52.419 16.730 1.000 13.31 ATOM 552 O TYR 73 −27.404 −52.630 15.764 1.000 10.40 ATOM 553 N LEU 74 −28.172 −53.261 17.759 1.000 8.99 ATOM 554 CA LEU 74 −27.204 −54.342 17.901 1.000 7.76 ATOM 555 CB LEU 74 −27.554 −55.155 19.155 1.000 9.47 ATOM 556 CG LEU 74 −26.402 −55.532 20.080 1.000 10.36 ATOM 557 CD1 LEU 74 −26.786 −56.729 20.939 1.000 25.33 ATOM 558 CD2 LEU 74 −25.137 −55.819 19.288 1.000 13.92 ATOM 559 C LEU 74 −27.088 −55.253 16.687 1.000 5.72 ATOM 560 O LEU 74 −25.980 −55.383 16.141 1.000 7.01 ATOM 561 N PRO 75 −28.141 −55.907 16.219 1.000 6.99 ATOM 562 CD PRO 75 −29.553 −55.794 16.615 1.000 1.55 ATOM 563 CA PRO 75 −27.965 −56.896 15.140 1.000 7.57 ATOM 564 CB PRO 75 −29.384 −57.401 14.855 1.000 5.01 ATOM 565 CG PRO 75 −30.158 −57.063 16.086 1.000 6.27 ATOM 566 C PRO 75 −27.364 −56.285 13.882 1.000 4.16 ATOM 567 O PRO 75 −26.651 −56.971 13.158 1.000 4.35 ATOM 568 N SER 76 −27.640 −55.014 13.615 1.000 6.22 ATOM 569 CA SER 76 −27.050 −54.322 12.473 1.000 0.00 ATOM 570 CB SER 76 −27.758 −52.978 12.261 1.000 0.00 ATOM 571 OG SER 76 −29.120 −53.249 11.920 1.000 0.00 ATOM 572 C SER 76 −25.554 −54.127 12.674 1.000 0.69 ATOM 573 O SER 76 −24.767 −54.280 11.740 1.000 4.06 ATOM 574 N CYS 77 −25.202 −53.802 13.911 1.000 2.82 ATOM 575 CA CYS 77 −23.851 −53.599 14.384 1.000 2.99 ATOM 576 CB CYS 77 −23.878 −53.202 15.868 1.000 0.00 ATOM 577 SG CYS 77 −22.325 −52.508 16.451 1.000 8.78 ATOM 578 C CYS 77 −22.962 −54.831 14.225 1.000 13.77 ATOM 579 O CYS 77 −21.828 −54.700 13.755 1.000 12.12 ATOM 580 N LEU 78 −23.455 −55.996 14.621 1.000 15.71 ATOM 581 CA LEU 78 −22.751 −57.268 14.538 1.000 10.13 ATOM 582 CB LEU 78 −23.617 −58.387 15.129 1.000 2.73 ATOM 583 CG LEU 78 −23.777 −58.354 16.651 1.000 7.98 ATOM 584 CD1 LEU 78 −24.866 −59.319 17.085 1.000 3.36 ATOM 585 CD2 LEU 78 −22.451 −58.676 17.330 1.000 8.53 ATOM 586 C LEU 78 −22.385 −57.650 13.106 1.000 9.88 ATOM 587 O LEU 78 −21.222 −57.855 12.761 1.000 12.55 ATOM 588 N ALA 79 −23.407 −57.748 12.271 1.000 11.93 ATOM 589 CA ALA 79 −23.297 −58.022 10.848 1.000 2.98 ATOM 590 CB ALA 79 −24.699 −58.042 10.255 1.000 0.32 ATOM 591 C ALA 79 −22.393 −57.026 10.127 1.000 7.73 ATOM 592 O ALA 79 −21.724 −57.408 9.163 1.000 13.15 ATOM 593 N THR 80 −22.337 −55.774 10.560 1.000 10.93 ATOM 594 CA THR 80 −21.427 −54.757 10.044 1.000 6.56 ATOM 595 CB THR 80 −21.703 −53.373 10.669 1.000 9.10 ATOM 596 OG1 THR 80 −23.013 −52.897 10.320 1.000 4.47 ATOM 597 CG2 THR 80 −20.722 −52.328 10.148 1.000 8.02 ATOM 598 C THR 80 −19.970 −55.117 10.317 1.000 10.87 ATOM 599 O THR 80 −19.103 −55.052 9.450 1.000 12.66 ATOM 600 N HIS 81 −19.659 −55.512 11.548 1.000 13.90 ATOM 601 CA HIS 81 −18.282 −55.720 11.978 1.000 13.04 ATOM 602 CB HIS 81 −18.119 −55.195 13.418 1.000 15.15 ATOM 603 CG HIS 81 −18.279 −53.704 13.502 1.000 10.10 ATOM 604 CD2 HIS 81 −19.202 −52.927 14.111 1.000 6.25 ATOM 605 ND1 HIS 81 −17.404 −52.833 12.889 1.000 7.20 ATOM 606 CE1 HIS 81 −17.775 −51.589 13.117 1.000 7.73 ATOM 607 NE2 HIS 81 −18.867 −51.616 13.863 1.000 6.24 ATOM 608 C HIS 81 −17.827 −57.166 11.896 1.000 9.61 ATOM 609 O HIS 81 −16.674 −57.460 12.216 1.000 10.35 ATOM 610 N LEU 82 −18.689 −58.081 11.470 1.000 4.74 ATOM 611 CA LEU 82 −18.257 −59.461 11.247 1.000 6.06 ATOM 612 CB LEU 82 −19.399 −60.263 10.631 1.000 6.90 ATOM 613 CG LEU 82 −20.535 −60.716 11.541 1.000 6.83 ATOM 614 CD1 LEU 82 −21.388 −61.774 10.851 1.000 11.79 ATOM 615 CD2 LEU 82 −19.987 −61.246 12.856 1.000 23.45 ATOM 616 C LEU 82 −17.042 −59.500 10.337 1.000 6.51 ATOM 617 O LEU 82 −16.972 −58.722 9.375 1.000 1.45 ATOM 618 N PRO 83 −16.056 −60.360 10.556 1.000 7.15 ATOM 619 CD PRO 83 −14.823 −60.374 9.731 1.000 0.00 ATOM 620 CA PRO 83 −16.043 −61.394 11.583 1.000 5.44 ATOM 621 CB PRO 83 −14.941 −62.341 11.067 1.000 9.33 ATOM 622 CG PRO 83 −13.968 −61.405 10.415 1.000 7.09 ATOM 623 C PRO 83 −15.638 −60.922 12.973 1.000 10.31 ATOM 624 O PRO 83 −14.716 −60.125 13.110 1.000 16.21 ATOM 625 N LEU 84 −16.319 −61.434 13.994 1.000 14.34 ATOM 626 CA LEU 84 −16.009 −61.132 15.382 1.000 10.66 ATOM 627 CB LEU 84 −17.165 −60.373 16.049 1.000 7.23 ATOM 628 CG LEU 84 −17.485 −59.010 15.434 1.000 2.01 ATOM 629 CD1 LEU 84 −18.843 −58.518 15.902 1.000 8.19 ATOM 630 CD2 LEU 84 −16.382 −58.019 15.766 1.000 5.93 ATOM 631 C LEU 84 −15.734 −62.386 16.203 1.000 7.34 ATOM 632 O LEU 84 −16.299 −63.447 15.945 1.000 8.40 ATOM 633 N ASP 85 −14.879 −62.247 17.208 1.000 8.68 ATOM 634 CA ASP 85 −14.607 −63.332 18.146 1.000 10.21 ATOM 635 CB ASP 85 −13.093 −63.433 18.382 1.000 15.96 ATOM 636 CG ASP 85 −12.338 −63.789 17.117 1.000 11.01 ATOM 637 OD1 ASP 85 −12.343 −64.975 16.727 1.000 9.49 ATOM 638 OD2 ASP 85 −11.739 −62.878 16.518 1.000 28.18 ATOM 639 C ASP 85 −15.313 −63.142 19.477 1.000 0.00 ATOM 640 O ASP 85 −15.778 −64.067 20.137 1.000 5.48 ATOM 641 N LEU 86 −15.414 −61.907 19.958 1.000 7.62 ATOM 642 CA LEU 86 −16.080 −61.695 21.243 1.000 8.84 ATOM 643 CB LEU 86 −15.085 −61.690 22.403 1.000 12.15 ATOM 644 CG LEU 86 −15.655 −61.580 23.822 1.000 13.98 ATOM 645 CD1 LEU 86 −16.562 −62.757 24.151 1.000 7.12 ATOM 646 CD2 LEU 86 −14.535 −61.477 24.850 1.000 10.28 ATOM 647 C LEU 86 −16.841 −60.374 21.221 1.000 6.69 ATOM 648 O LEU 86 −16.327 −59.409 20.649 1.000 8.05 ATOM 649 N VAL 87 −18.013 −60.361 21.842 1.000 4.26 ATOM 650 CA VAL 87 −18.752 −59.127 22.049 1.000 2.21 ATOM 651 CB VAL 87 −20.150 −59.126 21.413 1.000 8.44 ATOM 652 CG1 VAL 87 −20.848 −57.808 21.722 1.000 2.51 ATOM 653 CG2 VAL 87 −20.104 −59.352 19.911 1.000 0.00 ATOM 654 C VAL 87 −18.893 −58.869 23.551 1.000 7.05 ATOM 655 O VAL 87 −19.472 −59.660 24.289 1.000 5.76 ATOM 656 N ILE 88 −18.351 −57.746 24.010 1.000 7.24 ATOM 657 CA ILE 88 −18.499 −57.336 25.400 1.000 6.18 ATOM 658 CB ILE 88 −17.233 −56.652 25.938 1.000 6.54 ATOM 659 CG2 ILE 88 −17.458 −56.098 27.333 1.000 11.40 ATOM 660 CG1 ILE 88 −16.001 −57.559 25.902 1.000 6.21 ATOM 661 CD1 ILE 88 −14.734 −56.856 26.339 1.000 7.20 ATOM 662 C ILE 88 −19.693 −56.394 25.506 1.000 4.68 ATOM 663 O ILE 88 −19.817 −55.458 24.716 1.000 10.14 ATOM 664 N ILE 89 −20.574 −56.672 26.457 1.000 7.74 ATOM 665 CA ILE 89 −21.765 −55.857 26.645 1.000 12.20 ATOM 666 CB ILE 89 −23.052 −56.635 26.306 1.000 12.51 ATOM 667 CG2 ILE 89 −24.253 −55.703 26.339 1.000 11.52 ATOM 668 CG1 ILE 89 −22.981 −57.390 24.979 1.000 6.47 ATOM 669 CD1 ILE 89 −24.250 −58.111 24.597 1.000 8.71 ATOM 670 C ILE 89 −21.861 −55.340 28.078 1.000 11.05 ATOM 671 O ILE 89 −22.169 −56.106 28.989 1.000 3.02 ATOM 672 N MET 90 −21.590 −54.049 28.236 1.000 7.01 ATOM 673 CA MET 90 −21.808 −53.359 29.492 1.000 11.48 ATOM 674 CB MET 90 −20.535 −52.721 30.043 1.000 9.27 ATOM 675 CG MET 90 −20.756 −52.097 31.415 1.000 10.33 ATOM 676 XD MET 90 −19.202 −51.706 32.246 1.000 17.92 ATOM 677 CE MET 90 −18.544 −50.475 31.124 1.000 12.70 ATOM 678 C MET 90 −22.872 −52.262 29.325 1.000 12.90 ATOM 679 O MET 90 −22.524 −51.143 28.954 1.000 0.00 ATOM 680 N LEU 91 −24.108 −52.639 29.604 1.000 8.70 ATOM 681 CA LEU 91 −25.292 −51.802 29.511 1.000 10.58 ATOM 682 CB LEU 91 −26.114 −52.105 28.254 1.000 9.42 ATOM 683 CG LEU 91 −25.573 −51.564 26.932 1.000 4.10 ATOM 684 CD1 LEU 91 −26.427 −52.046 25.772 1.000 0.00 ATOM 685 CD2 LEU 91 −25.506 −50.044 26.961 1.000 2.02 ATOM 686 C LEU 91 −26.169 −52.031 30.734 1.000 2.21 ATOM 687 O LEU 91 −25.989 −53.066 31.388 1.000 10.59 ATOM 688 N GLY 92 −27.087 −51.117 31.025 1.000 4.69 ATOM 689 CA GLY 92 −27.963 −51.321 32.172 1.000 7.16 ATOM 690 C GLY 92 −28.189 −50.092 33.027 1.000 0.00 ATOM 691 O GLY 92 −29.266 −49.924 33.603 1.000 8.09 ATOM 692 N THR 93 −27.204 −49.219 33.133 1.000 0.16 ATOM 693 CA THR 93 −27.241 −48.005 33.929 1.000 9.42 ATOM 694 CB THR 93 −25.927 −47.205 33.768 1.000 17.05 ATOM 695 OG1 THR 93 −24.811 −48.063 34.024 1.000 26.81 ATOM 696 CG2 THR 93 −25.847 −46.068 34.778 1.000 0.34 ATOM 697 C THR 93 −28.386 −47.075 33.551 1.000 9.26 ATOM 698 O THR 93 −29.037 −46.491 34.419 1.000 14.18 ATOM 699 N ASN 94 −28.614 −46.927 32.250 1.000 0.69 ATOM 700 CA ASN 94 −29.609 −45.981 31.755 1.000 5.12 ATOM 701 CB ASN 94 −29.333 −45.677 30.274 1.000 9.42 ATOM 702 CG ASN 94 −27.990 −44.983 30.120 1.000 10.74 ATOM 703 OD1 ASN 94 −27.679 −44.062 30.873 1.000 21.66 ATOM 704 ND2 ASN 94 −27.175 −45.417 29.174 1.000 18.23 ATOM 705 C ASN 94 −31.029 −46.481 31.986 1.000 5.80 ATOM 706 O ASN 94 −31.889 −45.654 32.317 1.000 4.04 ATOM 707 N ASP 95 −31.282 −47.777 31.863 1.000 4.02 ATOM 708 CA ASP 95 −32.568 −48.411 32.137 1.000 7.86 ATOM 709 CB ASP 95 −32.522 −49.913 31.880 1.000 5.49 ATOM 710 CG ASP 95 −32.090 −50.392 30.521 1.000 10.09 ATOM 711 OD1 ASP 95 −30.998 −50.021 30.040 1.000 16.22 ATOM 712 OD2 ASP 95 −32.843 −51.184 29.907 1.000 15.98 ATOM 713 C ASP 95 −33.020 −48.208 33.591 1.000 9.17 ATOM 714 O ASP 95 −34.188 −48.361 33.958 1.000 0.43 ATOM 715 N THR 96 −32.051 −47.882 34.421 1.000 11.45 ATOM 716 CA THR 96 −32.122 −47.529 35.823 1.000 16.75 ATOM 717 CB THR 96 −30.697 −47.638 36.412 1.000 24.78 ATOM 718 OG1 THR 96 −30.607 −48.784 37.274 1.000 17.62 ATOM 719 CG2 THR 96 −30.350 −46.409 37.229 1.000 12.12 ATOM 720 C THR 96 −32.697 −46.132 35.997 1.000 12.12 ATOM 721 O THR 96 −33.047 −45.678 37.088 1.000 10.94 ATOM 722 N LYS 97 −32.820 −45.406 34.883 1.000 12.18 ATOM 723 CA LYS 97 −33.387 −44.060 34.954 1.000 14.27 ATOM 724 CB LYS 97 −33.247 −43.336 33.620 1.000 13.25 ATOM 725 CG LYS 97 −31.996 −42.477 33.500 1.000 11.50 ATOM 726 CD LYS 97 −31.819 −41.935 32.086 1.000 3.08 ATOM 727 CE LYS 97 −30.344 −41.856 31.717 1.000 0.00 ATOM 728 NZ LYS 97 −30.131 −41.152 30.416 1.000 0.00 ATOM 729 C LYS 97 −34.848 −44.112 35.403 1.000 12.44 ATOM 730 O LYS 97 −35.636 −44.914 34.911 1.000 8.04 ATOM 731 N ALA 98 −35.179 −43.246 36.355 1.000 11.97 ATOM 732 CA ALA 98 −36.454 −43.218 37.047 1.000 4.97 ATOM 733 CB ALA 98 −36.522 −41.982 37.943 1.000 3.36 ATOM 734 C ALA 98 −37.641 −43.246 36.100 1.000 12.00 ATOM 735 O ALA 98 −38.651 −43.905 36.355 1.000 22.61 ATOM 736 N TYR 99 −37.535 −42.518 34.988 1.000 12.39 ATOM 737 CA TYR 99 −38.695 −42.403 34.107 1.000 7.25 ATOM 738 CB TYR 99 −38.521 −41.297 33.087 1.000 9.11 ATOM 739 CG TYR 99 −37.300 −41.251 32.217 1.000 15.58 ATOM 740 CD1 TYR 99 −37.261 −41.912 30.995 1.000 13.09 ATOM 741 CE1 TYR 99 −36.144 −41.874 30.186 1.000 9.06 ATOM 742 CD2 TYR 99 −36.173 −40.533 32.598 1.000 14.48 ATOM 743 CE2 TYR 99 −35.051 −40.482 31.796 1.000 15.13 ATOM 744 CZ TYR 99 −35.044 −41.154 30.591 1.000 11.74 ATOM 745 OH TYR 99 −33.925 −41.102 29.794 1.000 6.20 ATOM 746 C TYR 99 −38.990 −43.726 33.413 1.000 11.25 ATOM 747 O TYR 99 −40.121 −43.927 32.963 1.000 12.89 ATOM 748 N PHE 100 −37.993 −44.606 33.351 1.000 4.63 ATOM 749 CA PHE 100 −38.237 −45.908 32.731 1.000 1.01 ATOM 750 CB PHE 100 −36.903 −46.556 32.348 1.000 3.41 ATOM 751 CG PHE 100 −36.316 −45.980 31.070 1.000 11.77 ATOM 752 CD1 PHE 100 −35.018 −45.506 31.032 1.000 7.50 ATOM 753 CD2 PHE 100 −37.080 −45.919 29.917 1.000 16.94 ATOM 754 CE1 PHE 100 −34.489 −44.981 29.868 1.000 7.31 ATOM 755 CE2 PHE 100 −36.557 −45.398 28.748 1.000 12.92 ATOM 756 CZ PHE 100 −35.260 −44.925 28.722 1.000 7.58 ATOM 757 C PHE 100 −39.051 −46.829 33.628 1.000 6.94 ATOM 758 O PHE 100 −39.711 −47.750 33.131 1.000 9.31 ATOM 759 N ARG 101 −39.032 −46.629 34.943 1.000 12.10 ATOM 760 CA ARG 101 −39.783 −47.468 35.869 1.000 12.96 ATOM 761 CB ARG 101 −41.294 −47.296 35.695 1.000 16.21 ATOM 762 CG ARG 101 −41.890 −45.959 36.087 1.000 19.51 ATOM 763 CD ARG 101 −43.376 −45.918 35.740 1.000 25.82 ATOM 764 NE ARG 101 −43.818 −44.553 35.466 1.000 31.88 ATOM 765 CZ ARG 101 −43.797 −43.583 36.373 1.000 33.97 ATOM 766 NH1 ARG 101 −43.355 −43.839 37.599 1.000 43.49 ATOM 767 NH2 ARG 101 −44.206 −42.361 36.067 1.000 44.85 ATOM 768 C ARG 101 −39.472 −48.955 35.704 1.000 12.20 ATOM 769 O ARG 101 −40.376 −49.782 35.878 1.000 12.48 ATOM 770 N ARG 102 −38.238 −49.319 35.378 1.000 8.86 ATOM 771 CA ARG 102 −37.887 −50.733 35.264 1.000 11.00 ATOM 772 CB ARG 102 −36.899 −50.962 34.115 1.000 6.96 ATOM 773 CG ARG 102 −37.497 −50.805 32.720 1.000 9.64 ATOM 774 CD ARG 102 −36.518 −51.198 31.624 1.000 8.07 ATOM 775 NE ARG 102 −37.140 −51.842 30.474 1.000 4.64 ATOM 776 CZ ARG 102 −36.540 −52.606 29.571 1.000 7.34 ATOM 777 NH1 ARG 102 −35.240 −52.877 29.628 1.000 1.45 ATOM 778 NH2 ARG 102 −37.232 −53.131 28.567 1.000 6.11 ATOM 779 C ARG 102 −37.320 −51.275 36.577 1.000 11.09 ATOM 780 O ARG 102 −36.734 −50.567 37.394 1.000 10.02 ATOM 781 N THR 103 −37.497 −52.573 36.785 1.000 11.01 ATOM 782 CA THR 103 −36.898 −53.307 37.893 1.000 12.65 ATOM 783 CB THR 103 −37.844 −54.376 38.462 1.000 7.64 ATOM 784 OG1 THR 103 −38.083 −55.384 37.468 1.000 11.29 ATOM 785 CG2 THR 103 −39.199 −53.771 38.790 1.000 15.33 ATOM 786 C THR 103 −35.618 −53.966 37.390 1.000 10.55 ATOM 787 O THR 103 −35.409 −53.986 36.173 1.000 9.17 ATOM 788 N PRO 104 −34.765 −54.474 38.264 1.000 10.17 ATOM 789 CD PRO 104 −34.799 −54.363 39.731 1.000 14.03 ATOM 790 CA PRO 104 −33.598 −55.230 37.803 1.000 6.81 ATOM 791 CB PRO 104 −32.968 −55.748 39.094 1.000 5.25 ATOM 792 CG PRO 104 −33.402 −54.759 40.129 1.000 8.07 ATOM 793 C PRO 104 −34.010 −56.400 36.911 1.000 5.89 ATOM 794 O PRO 104 −33.251 −56.728 35.998 1.000 5.49 ATOM 795 N LEU 105 −35.164 −56.994 37.173 1.000 2.55 ATOM 796 CA LEU 105 −35.690 −58.071 36.341 1.000 10.27 ATOM 797 CB LEU 105 −36.989 −58.642 36.890 1.000 11.51 ATOM 798 CG LEU 105 −37.304 −60.122 36.695 1.000 16.39 ATOM 799 CD1 LEU 105 −38.804 −60.319 36.480 1.000 4.05 ATOM 800 CD2 LEU 105 −36.533 −60.744 35.542 1.000 15.49 ATOM 801 C LEU 105 −35.923 −57.566 34.915 1.000 14.30 ATOM 802 O LEU 105 −35.415 −58.168 33.969 1.000 14.22 ATOM 803 N ASP 106 −36.686 −56.484 34.791 1.000 11.11 ATOM 804 CA ASP 106 −36.922 −55.878 33.482 1.000 8.08 ATOM 805 CB ASP 106 −37.636 −54.538 33.621 1.000 14.02 ATOM 806 CG ASP 106 −39.046 −54.638 34.152 1.000 13.88 ATOM 807 OD1 ASP 106 −39.726 −55.653 33.875 1.000 19.94 ATOM 808 OD2 ASP 106 −39.479 −53.686 34.843 1.000 4.29 ATOM 809 C ASP 106 −35.607 −55.668 32.734 1.000 7.79 ATOM 810 O ASP 106 −35.504 −55.987 31.554 1.000 10.52 ATOM 811 N ILE 107 −34.614 −55.131 33.438 1.000 5.00 ATOM 812 CA ILE 107 −33.321 −54.814 32.845 1.000 6.63 ATOM 813 CB ILE 107 −32.444 −54.016 33.828 1.000 14.49 ATOM 814 CG2 ILE 107 −31.125 −53.622 33.184 1.000 7.24 ATOM 815 CG1 ILE 107 −33.146 −52.790 34.415 1.000 16.93 ATOM 816 CD1 ILE 107 −32.174 −51.779 34.992 1.000 19.38 ATOM 817 C ILE 107 −32.564 −56.059 32.405 1.000 5.12 ATOM 818 O ILE 107 −31.877 −56.024 31.381 1.000 4.80 ATOM 819 N ALA 108 −32.691 −57.148 33.157 1.000 5.34 ATOM 820 CA ALA 108 −32.021 −58.398 32.812 1.000 4.25 ATOM 821 CB ALA 108 −32.089 −59.399 33.956 1.000 2.49 ATOM 822 C ALA 108 −32.637 −59.018 31.568 1.000 2.89 ATOM 823 O ALA 108 −31.952 −59.619 30.738 1.000 11.68 ATOM 824 N LEU 109 −33.956 −58.864 31.449 1.000 0.00 ATOM 825 CA LEU 109 −34.609 −59.401 30.251 1.000 6.18 ATOM 826 CB LEU 109 −36.125 −59.391 30.435 1.000 12.37 ATOM 827 CG LEU 109 −36.674 −60.463 31.386 1.000 15.66 ATOM 828 CD1 LEU 109 −37.985 −60.004 32.001 1.000 27.44 ATOM 829 CD2 LEU 109 −36.854 −61.794 30.672 1.000 3.14 ATOM 830 C LEU 109 −34.171 −58.620 29.022 1.000 10.30 ATOM 831 O LEU 109 −34.035 −59.139 27.915 1.000 18.00 ATOM 832 N GLY 110 −33.918 −57.323 29.193 1.000 11.78 ATOM 833 CA GLY 110 −33.426 −56.535 28.069 1.000 8.26 ATOM 834 C GLY 110 −32.028 −56.976 27.666 1.000 7.06 ATOM 835 O GLY 110 −31.757 −57.155 26.482 1.000 18.68 ATOM 836 N MET 111 −31.149 −57.149 28.651 1.000 5.04 ATOM 837 CA MET 111 −29.812 −57.661 28.414 1.000 4.52 ATOM 838 CB MET 111 −28.962 −57.717 29.683 1.000 1.61 ATOM 839 CG MET 111 −27.663 −58.503 29.542 1.000 0.00 ATOM 840 XD MET 111 −26.456 −57.694 28.453 1.000 16.83 ATOM 841 CE MET 111 −25.895 −56.355 29.497 1.000 5.08 ATOM 842 C MET 111 −29.915 −59.066 27.821 1.000 6.40 ATOM 843 O MET 111 −29.098 −59.476 27.005 1.000 8.66 ATOM 844 N SER 112 −30.937 −59.795 28.270 1.000 9.55 ATOM 845 CA SER 112 −31.140 −61.133 27.731 1.000 8.05 ATOM 846 CB SER 112 −32.322 −61.821 28.405 1.000 10.37 ATOM 847 OG SER 112 −33.488 −61.744 27.609 1.000 8.11 ATOM 848 C SER 112 −31.341 −61.034 26.217 1.000 6.07 ATOM 849 O SER 112 −30.761 −61.823 25.471 1.000 9.26 ATOM 850 N VAL 113 −32.142 −60.065 25.803 1.000 4.80 ATOM 851 CA VAL 113 −32.424 −59.788 24.401 1.000 9.22 ATOM 852 CB VAL 113 −33.414 −58.615 24.266 1.000 9.35 ATOM 853 CG1 VAL 113 −33.350 −57.979 22.886 1.000 0.53 ATOM 854 CG2 VAL 113 −34.830 −59.090 24.567 1.000 15.43 ATOM 855 C VAL 113 −31.149 −59.490 23.616 1.000 18.19 ATOM 856 O VAL 113 −31.027 −59.900 22.456 1.000 17.08 ATOM 857 N LEU 114 −30.199 −58.791 24.235 1.000 16.22 ATOM 858 CA LEU 114 −28.948 −58.431 23.570 1.000 9.05 ATOM 859 CB LEU 114 −28.220 −57.329 24.341 1.000 4.93 ATOM 860 CG LEU 114 −28.938 −55.983 24.427 1.000 6.23 ATOM 861 CD1 LEU 114 −28.122 −54.973 25.221 1.000 8.47 ATOM 862 CD2 LEU 114 −29.228 −55.450 23.032 1.000 0.00 ATOM 863 C LEU 114 −28.018 −59.628 23.407 1.000 5.15 ATOM 864 O LEU 114 −27.310 −59.762 22.410 1.000 8.05 ATOM 865 N VAL 115 −28.028 −60.503 24.403 1.000 5.78 ATOM 866 CA VAL 115 −27.223 −61.717 24.373 1.000 8.93 ATOM 867 CB VAL 115 −27.202 −62.383 25.762 1.000 8.05 ATOM 868 CG1 VAL 115 −26.501 −63.729 25.720 1.000 0.00 ATOM 869 CG2 VAL 115 −26.543 −61.439 26.759 1.000 0.00 ATOM 870 C VAL 115 −27.763 −62.685 23.330 1.000 9.50 ATOM 871 O VAL 115 −27.007 −63.390 22.662 1.000 9.58 ATOM 872 N THR 116 −29.087 −62.715 23.179 1.000 8.15 ATOM 873 CA THR 116 −29.688 −63.617 22.199 1.000 8.38 ATOM 874 CB THR 116 −31.222 −63.622 22.327 1.000 12.50 ATOM 875 OG1 THR 116 −31.575 −64.207 23.585 1.000 13.40 ATOM 876 CG2 THR 116 −31.848 −64.479 21.233 1.000 10.82 ATOM 877 C THR 116 −29.316 −63.241 20.771 1.000 5.56 ATOM 878 O THR 116 −29.011 −64.127 19.966 1.000 5.27 ATOM 879 N GLN 117 −29.345 −61.945 20.473 1.000 8.17 ATOM 880 CA GLN 117 −28.956 −61.430 19.160 1.000 9.93 ATOM 881 CB GLN 117 −29.166 −59.920 19.080 1.000 3.66 ATOM 882 CG GLN 117 −30.592 −59.440 19.279 1.000 6.21 ATOM 883 CD GLN 117 −30.699 −57.933 19.390 1.000 7.09 ATOM 884 OE1 GLN 117 −29.801 −57.260 19.896 1.000 12.85 ATOM 885 NE2 GLN 117 −31.811 −57.376 18.914 1.000 7.39 ATOM 886 C GLN 117 −27.499 −61.761 18.847 1.000 11.60 ATOM 887 O GLN 117 −27.105 −62.023 17.706 1.000 9.03 ATOM 888 N VAL 118 −26.652 −61.751 19.879 1.000 11.77 ATOM 889 CA VAL 118 −25.258 −62.146 19.659 1.000 8.34 ATOM 890 CB VAL 118 −24.340 −61.768 20.831 1.000 0.49 ATOM 891 CG1 VAL 118 −22.892 −62.118 20.499 1.000 21.94 ATOM 892 CG2 VAL 118 −24.452 −60.291 21.169 1.000 3.31 ATOM 893 C VAL 118 −25.166 −63.652 19.417 1.000 10.48 ATOM 894 O VAL 118 −24.354 −64.107 18.607 1.000 10.54 ATOM 895 N LEU 119 −25.993 −64.431 20.112 1.000 7.97 ATOM 896 CA LEU 119 −25.916 −65.885 19.993 1.000 8.73 ATOM 897 CB LEU 119 −26.679 −66.572 21.135 1.000 8.06 ATOM 898 CG LEU 119 −25.981 −66.556 22.498 1.000 21.06 ATOM 899 CD1 LEU 119 −26.800 −67.296 23.548 1.000 5.53 ATOM 900 CD2 LEU 119 −24.580 −67.150 22.403 1.000 21.96 ATOM 901 C LEU 119 −26.446 −66.362 18.649 1.000 5.78 ATOM 902 O LEU 119 −26.022 −67.409 18.153 1.000 14.06 ATOM 903 N THR 120 −27.364 −65.608 18.053 1.000 8.82 ATOM 904 CA THR 120 −27.964 −65.985 16.780 1.000 0.00 ATOM 905 CB THR 120 −29.497 −65.798 16.815 1.000 6.15 ATOM 906 OG1 THR 120 −29.805 −64.405 16.969 1.000 10.14 ATOM 907 CG2 THR 120 −30.121 −66.535 17.994 1.000 0.76 ATOM 908 C THR 120 −27.419 −65.198 15.594 1.000 10.30 ATOM 909 O THR 120 −28.061 −65.190 14.537 1.000 13.46 ATOM 910 N SER 121 −26.272 −64.533 15.700 1.000 11.26 ATOM 911 CA SER 121 −25.774 −63.675 14.636 1.000 7.70 ATOM 912 CB SER 121 −25.000 −62.487 15.240 1.000 5.36 ATOM 913 OG SER 121 −23.826 −62.954 15.886 1.000 3.70 ATOM 914 C SER 121 −24.852 −64.353 13.629 1.000 7.89 ATOM 915 O SER 121 −24.360 −63.660 12.730 1.000 13.24 ATOM 916 N ALA 122 −24.603 −65.645 13.755 1.000 11.50 ATOM 917 CA ALA 122 −23.748 −66.370 12.820 1.000 12.48 ATOM 918 CB ALA 122 −23.820 −67.868 13.098 1.000 3.73 ATOM 919 C ALA 122 −24.124 −66.083 11.370 1.000 7.92 ATOM 920 O ALA 122 −25.311 −66.050 11.042 1.000 8.42 ATOM 921 N GLY 123 −23.125 −65.859 10.529 1.000 7.14 ATOM 922 CA GLY 123 −23.316 −65.625 9.115 1.000 3.98 ATOM 923 C GLY 123 −23.643 −64.196 8.735 1.000 12.34 ATOM 924 O GLY 123 −23.445 −63.822 7.571 1.000 1.55 ATOM 925 N GLY 124 −24.132 −63.404 9.683 1.000 19.09 ATOM 926 CA GLY 124 −24.506 −62.016 9.471 1.000 13.26 ATOM 927 C GLY 124 −25.277 −61.809 8.186 1.000 10.25 ATOM 928 O GLY 124 −26.403 −62.278 8.018 1.000 10.97 ATOM 929 N VAL 125 −24.684 −61.110 7.217 1.000 12.50 ATOM 930 CA VAL 125 −25.365 −60.956 5.930 1.000 9.40 ATOM 931 CB VAL 125 −25.557 −59.477 5.559 1.000 14.11 ATOM 932 CG1 VAL 125 −26.156 −59.326 4.168 1.000 13.51 ATOM 933 CG2 VAL 125 −26.455 −58.786 6.578 1.000 22.31 ATOM 934 C VAL 125 −24.588 −61.675 4.833 1.000 6.71 ATOM 935 O VAL 125 −23.580 −61.151 4.368 1.000 4.54 ATOM 936 N GLY 126 −25.047 −62.850 4.427 1.000 14.20 ATOM 937 CA GLY 126 −24.466 −63.654 3.377 1.000 9.15 ATOM 938 C GLY 126 −23.012 −64.018 3.580 1.000 10.06 ATOM 939 O GLY 126 −22.225 −64.068 2.629 1.000 4.29 ATOM 940 N THR 127 −22.595 −64.295 4.811 1.000 6.29 ATOM 941 CA THR 127 −21.214 −64.701 5.050 1.000 3.83 ATOM 942 CB THR 127 −20.470 −63.707 5.957 1.000 8.35 ATOM 943 OG1 THR 127 −20.719 −64.001 7.339 1.000 16.55 ATOM 944 CG2 THR 127 −20.987 −62.295 5.716 1.000 11.34 ATOM 945 C THR 127 −21.143 −66.099 5.663 1.000 1.10 ATOM 946 O THR 127 −22.159 −66.699 6.001 1.000 4.52 ATOM 947 N THR 128 −19.921 −66.590 5.790 1.000 9.21 ATOM 948 CA THR 128 −19.546 −67.893 6.299 1.000 8.72 ATOM 949 CB THR 128 −18.451 −68.505 5.397 1.000 10.99 ATOM 950 OG1 THR 128 −17.447 −67.497 5.236 1.000 7.85 ATOM 951 CG2 THR 128 −18.976 −68.853 4.015 1.000 3.45 ATOM 952 C THR 128 −18.995 −67.821 7.718 1.000 13.03 ATOM 953 O THR 128 −18.450 −68.788 8.255 1.000 8.50 ATOM 954 N TYR 129 −19.127 −66.646 8.315 1.000 10.20 ATOM 955 CA TYR 129 −18.542 −66.357 9.615 1.000 7.58 ATOM 956 CB TYR 129 −18.323 −64.853 9.722 1.000 8.22 ATOM 957 CG TYR 129 −17.246 −64.280 8.835 1.000 11.97 ATOM 958 CD1 TYR 129 −17.514 −63.176 8.031 1.000 8.62 ATOM 959 CE1 TYR 129 −16.547 −62.636 7.211 1.000 7.23 ATOM 960 CD2 TYR 129 −15.970 −64.827 8.799 1.000 12.10 ATOM 961 CE2 TYR 129 −14.991 −64.290 7.982 1.000 16.92 ATOM 962 CZ TYR 129 −15.288 −63.196 7.193 1.000 16.10 ATOM 963 OH TYR 129 −14.315 −62.655 6.383 1.000 11.56 ATOM 964 C TYR 129 −19.416 −66.840 10.765 1.000 9.63 ATOM 965 O TYR 129 −20.644 −66.723 10.714 1.000 13.75 ATOM 966 N PRO 130 −18.789 −67.380 11.804 1.000 8.51 ATOM 967 CD PRO 130 −17.336 −67.523 12.004 1.000 10.11 ATOM 968 CA PRO 130 −19.549 −67.914 12.938 1.000 5.53 ATOM 969 CB PRO 130 −18.522 −68.804 13.647 1.000 8.51 ATOM 970 CG PRO 130 −17.227 −68.097 13.397 1.000 11.17 ATOM 971 C PRO 130 −19.983 −66.791 13.872 1.000 7.77 ATOM 972 O PRO 130 −19.500 −65.667 13.730 1.000 2.72 ATOM 973 N ALA 131 −20.873 −67.117 14.799 1.000 7.61 ATOM 974 CA ALA 131 −21.305 −66.205 15.844 1.000 2.73 ATOM 975 CB ALA 131 −22.537 −66.747 16.554 1.000 0.00 ATOM 976 C ALA 131 −20.174 −65.984 16.842 1.000 8.30 ATOM 977 O ALA 131 −19.502 −66.942 17.223 1.000 12.18 ATOM 978 N PRO 132 −19.937 −64.752 17.273 1.000 14.28 ATOM 979 CD PRO 132 −20.610 −63.516 16.842 1.000 11.04 ATOM 980 CA PRO 132 −18.901 −64.505 18.284 1.000 12.37 ATOM 981 CB PRO 132 −18.696 −62.992 18.181 1.000 14.35 ATOM 982 CG PRO 132 −20.032 −62.472 17.753 1.000 12.70 ATOM 983 C PRO 132 −19.395 −64.884 19.675 1.000 12.80 ATOM 984 O PRO 132 −20.608 −65.027 19.856 1.000 21.24 ATOM 985 N LYS 133 −18.497 −65.051 20.641 1.000 14.17 ATOM 986 CA LYS 133 −18.903 −65.337 22.017 1.000 14.31 ATOM 987 CB LYS 133 −17.760 −65.881 22.869 1.000 14.22 ATOM 988 CG LYS 133 −17.050 −67.101 22.317 1.000 13.51 ATOM 989 CD LYS 133 −15.746 −67.358 23.057 1.000 18.76 ATOM 990 CE LYS 133 −15.463 −68.849 23.174 1.000 21.23 ATOM 991 NZ LYS 133 −15.154 −69.237 24.580 1.000 37.08 ATOM 992 C LYS 133 −19.441 −64.066 22.667 1.000 10.23 ATOM 993 O LYS 133 −19.319 −62.982 22.091 1.000 4.45 ATOM 994 N VAL 134 −20.032 −64.194 23.853 1.000 4.74 ATOM 995 CA VAL 134 −20.562 −63.000 24.507 1.000 10.55 ATOM 996 CB VAL 134 −22.106 −62.964 24.490 1.000 11.86 ATOM 997 CG1 VAL 134 −22.586 −61.523 24.423 1.000 0.00 ATOM 998 CG2 VAL 134 −22.659 −63.778 23.334 1.000 29.88 ATOM 999 C VAL 134 −20.129 −62.885 25.963 1.000 12.01 ATOM 1000 O VAL 134 −20.215 −63.837 26.736 1.000 27.94 ATOM 1001 N LEU 135 −19.676 −61.703 26.357 1.000 12.21 ATOM 1002 CA LEU 135 −19.364 −61.443 27.757 1.000 14.41 ATOM 1003 CB LEU 135 −17.975 −60.835 27.898 1.000 17.37 ATOM 1004 CG LEU 135 −17.123 −61.223 29.105 1.000 18.57 ATOM 1005 CD1 LEU 135 −15.993 −60.213 29.264 1.000 4.42 ATOM 1006 CD2 LEU 135 −17.932 −61.341 30.387 1.000 6.01 ATOM 1007 C LEU 135 −20.397 −60.497 28.360 1.000 17.03 ATOM 1008 O LEU 135 −20.485 −59.326 27.984 1.000 14.19 ATOM 1009 N VAL 136 −21.196 −60.988 29.303 1.000 19.10 ATOM 1010 CA VAL 136 −22.167 −60.110 29.954 1.000 14.45 ATOM 1011 CB VAL 136 −23.344 −60.925 30.511 1.000 13.65 ATOM 1012 CG1 VAL 136 −24.272 −60.045 31.335 1.000 8.06 ATOM 1013 CG2 VAL 136 −24.080 −61.596 29.362 1.000 0.00 ATOM 1014 C VAL 136 −21.498 −59.327 31.073 1.000 10.63 ATOM 1015 O VAL 136 −20.929 −59.948 31.971 1.000 7.12 ATOM 1016 N VAL 137 −21.556 −57.997 31.027 1.000 7.93 ATOM 1017 CA VAL 137 −20.882 −57.215 32.056 1.000 6.63 ATOM 1018 CB VAL 137 −19.699 −56.397 31.497 1.000 6.08 ATOM 1019 CG1 VAL 137 −19.115 −55.512 32.595 1.000 6.59 ATOM 1020 CG2 VAL 137 −18.609 −57.291 30.936 1.000 10.34 ATOM 1021 C VAL 137 −21.828 −56.255 32.775 1.000 6.02 ATOM 1022 O VAL 137 −22.319 −55.273 32.219 1.000 11.10 ATOM 1023 N SER 138 −22.061 −56.558 34.040 1.000 6.05 ATOM 1024 CA SER 138 −22.800 −55.715 34.972 1.000 9.77 ATOM 1025 CB SER 138 −23.139 −56.523 36.223 1.000 16.98 ATOM 1026 OG SER 138 −23.850 −55.804 37.202 1.000 19.18 ATOM 1027 C SER 138 −21.944 −54.496 35.276 1.000 8.41 ATOM 1028 O SER 138 −20.779 −54.646 35.652 1.000 13.52 ATOM 1029 N PRO 139 −22.459 −53.287 35.096 1.000 12.22 ATOM 1030 CD PRO 139 −23.803 −52.952 34.599 1.000 11.54 ATOM 1031 CA PRO 139 −21.657 −52.087 35.389 1.000 6.14 ATOM 1032 CB PRO 139 −22.422 −51.015 34.608 1.000 7.78 ATOM 1033 CG PRO 139 −23.848 −51.455 34.731 1.000 3.74 ATOM 1034 C PRO 139 −21.620 −51.775 36.875 1.000 3.92 ATOM 1035 O PRO 139 −22.460 −52.217 37.664 1.000 10.47 ATOM 1036 N PRO 140 −20.636 −51.014 37.347 1.000 8.52 ATOM 1037 CD PRO 140 −19.524 −50.412 36.611 1.000 3.33 ATOM 1038 CA PRO 140 −20.591 −50.724 38.788 1.000 13.50 ATOM 1039 CB PRO 140 −19.251 −50.012 38.971 1.000 12.27 ATOM 1040 CG PRO 140 −18.843 −49.543 37.623 1.000 6.73 ATOM 1041 C PRO 140 −21.748 −49.832 39.228 1.000 15.77 ATOM 1042 O PRO 140 −22.321 −49.073 38.445 1.000 21.96 ATOM 1043 N PRO 141 −22.103 −49.939 40.505 1.000 4.93 ATOM 1044 CD PRO 141 −21.487 −50.799 41.528 1.000 0.26 ATOM 1045 CA PRO 141 −23.230 −49.172 41.036 1.000 3.17 ATOM 1046 CB PRO 141 −23.254 −49.560 42.521 1.000 4.18 ATOM 1047 CG PRO 141 −22.591 −50.897 42.556 1.000 0.00 ATOM 1048 C PRO 141 −23.014 −47.671 40.890 1.000 10.32 ATOM 1049 O PRO 141 −21.876 −47.203 40.900 1.000 17.58 ATOM 1050 N LEU 142 −24.120 −46.942 40.760 1.000 9.20 ATOM 1051 CA LEU 142 −24.079 −45.490 40.729 1.000 7.44 ATOM 1052 CB LEU 142 −25.421 −44.900 40.288 1.000 7.55 ATOM 1053 CG LEU 142 −25.775 −45.119 38.812 1.000 13.23 ATOM 1054 CD1 LEU 142 −27.262 −44.901 38.566 1.000 0.00 ATOM 1055 CD2 LEU 142 −24.932 −44.218 37.921 1.000 1.85 ATOM 1056 C LEU 142 −23.711 −44.945 42.109 1.000 13.38 ATOM 1057 O LEU 142 −23.764 −45.680 43.099 1.000 20.55 ATOM 1058 N ALA 143 −23.363 −43.670 42.126 1.000 15.81 ATOM 1059 CA ALA 143 −22.960 −42.941 43.322 1.000 13.69 ATOM 1060 CB ALA 143 −21.461 −42.676 43.239 1.000 3.16 ATOM 1061 C ALA 143 −23.762 −41.656 43.475 1.000 16.69 ATOM 1062 O ALA 143 −24.500 −41.280 42.552 1.000 10.61 ATOM 1063 N PRO 144 −23.668 −40.968 44.609 1.000 19.19 ATOM 1064 CD PRO 144 −22.997 −41.377 45.852 1.000 16.93 ATOM 1065 CA PRO 144 −24.315 −39.659 44.745 1.000 19.29 ATOM 1066 CB PRO 144 −23.730 −39.076 46.031 1.000 17.13 ATOM 1067 CG PRO 144 −22.904 −40.130 46.664 1.000 12.97 ATOM 1068 C PRO 144 −24.009 −38.723 43.578 1.000 17.14 ATOM 1069 O PRO 144 −22.902 −38.626 43.048 1.000 12.89 ATOM 1070 N MET 145 −25.049 −38.002 43.161 1.000 18.09 ATOM 1071 CA MET 145 −24.925 −37.064 42.052 1.000 14.70 ATOM 1072 CB MET 145 −25.912 −37.398 40.942 1.000 21.06 ATOM 1073 CG MET 145 −25.711 −38.740 40.263 1.000 24.88 ATOM 1074 XD MET 145 −27.259 −39.577 39.860 1.000 18.47 ATOM 1075 CE MET 145 −27.956 −39.804 41.495 1.000 34.91 ATOM 1076 C MET 145 −25.155 −35.645 42.559 1.000 11.49 ATOM 1077 O MET 145 −26.205 −35.342 43.116 1.000 18.46 ATOM 1078 N PRO 146 −24.182 −34.763 42.367 1.000 6.41 ATOM 1079 CD PRO 146 −22.909 −34.993 41.683 1.000 8.62 ATOM 1080 CA PRO 146 −24.325 −33.388 42.851 1.000 10.88 ATOM 1081 CB PRO 146 −22.916 −32.814 42.759 1.000 10.59 ATOM 1082 CG PRO 146 −22.064 −33.819 42.072 1.000 12.17 ATOM 1083 C PRO 146 −25.292 −32.588 41.972 1.000 13.13 ATOM 1084 O PRO 146 −25.999 −31.712 42.484 1.000 17.39 ATOM 1085 N HIS 147 −25.311 −32.901 40.677 1.000 10.50 ATOM 1086 CA HIS 147 −26.203 −32.215 39.758 1.000 9.69 ATOM 1087 CB HIS 147 −25.865 −32.480 38.279 1.000 14.24 ATOM 1088 CG HIS 147 −26.441 −31.373 37.431 1.000 6.69 ATOM 1089 CD2 HIS 147 −25.875 −30.297 36.850 1.000 5.99 ATOM 1090 ND1 HIS 147 −27.780 −31.296 37.134 1.000 11.40 ATOM 1091 CE1 HIS 147 −28.018 −30.226 36.391 1.000 11.68 ATOM 1092 NE2 HIS 147 −26.871 −29.600 36.201 1.000 12.68 ATOM 1093 C HIS 147 −27.658 −32.596 40.013 1.000 5.47 ATOM 1094 O HIS 147 −28.052 −33.761 39.960 1.000 11.15 ATOM 1095 N PRO 148 −28.463 −31.575 40.291 1.000 12.88 ATOM 1096 CD PRO 148 −28.098 −30.148 40.322 1.000 12.98 ATOM 1097 CA PRO 148 −29.877 −31.806 40.602 1.000 13.30 ATOM 1098 CB PRO 148 −30.440 −30.401 40.811 1.000 14.82 ATOM 1099 CG PRO 148 −29.426 −29.455 40.267 1.000 16.64 ATOM 1100 C PRO 148 −30.600 −32.508 39.456 1.000 15.39 ATOM 1101 O PRO 148 −31.525 −33.290 39.689 1.000 15.71 ATOM 1102 N TRP 149 −30.218 −32.263 38.201 1.000 21.29 ATOM 1103 CA TRP 149 −30.909 −32.947 37.109 1.000 15.64 ATOM 1104 CB TRP 149 −30.571 −32.328 35.750 1.000 17.31 ATOM 1105 CG TRP 149 −31.296 −33.043 34.639 1.000 10.06 ATOM 1106 CD2 TRP 149 −32.715 −33.086 34.444 1.000 4.30 ATOM 1107 CE2 TRP 149 −32.952 −33.862 33.295 1.000 8.55 ATOM 1108 CE3 TRP 149 −33.805 −32.541 35.129 1.000 4.24 ATOM 1109 CD1 TRP 149 −30.748 −33.774 33.629 1.000 11.09 ATOM 1110 NE1 TRP 149 −31.736 −34.272 32.813 1.000 5.61 ATOM 1111 CZ2 TRP 149 −34.240 −34.107 32.815 1.000 12.36 ATOM 1112 CZ3 TRP 149 −35.076 −32.785 34.654 1.000 13.41 ATOM 1113 CH2 TRP 149 −35.286 −33.563 33.505 1.000 14.13 ATOM 1114 C TRP 149 −30.566 −34.432 37.101 1.000 12.85 ATOM 1115 O TRP 149 −31.447 −35.290 37.033 1.000 7.92 ATOM 1116 N PHE 150 −29.270 −34.728 37.186 1.000 11.11 ATOM 1117 CA PHE 150 −28.841 −36.125 37.305 1.000 11.76 ATOM 1118 CB PHE 150 −27.321 −36.192 37.483 1.000 8.65 ATOM 1119 CG PHE 150 −26.581 −36.170 36.150 1.000 13.44 ATOM 1120 CD1 PHE 150 −25.315 −35.623 36.047 1.000 14.41 ATOM 1121 CD2 PHE 150 −27.167 −36.697 35.014 1.000 12.01 ATOM 1122 CE1 PHE 150 −24.650 −35.604 34.838 1.000 14.96 ATOM 1123 CE2 PHE 150 −26.511 −36.684 33.797 1.000 13.41 ATOM 1124 CZ PHE 150 −25.246 −36.136 33.711 1.000 18.95 ATOM 1125 C PHE 150 −29.555 −36.813 38.459 1.000 10.90 ATOM 1126 O PHE 150 −30.059 −37.930 38.354 1.000 7.95 ATOM 1127 N GLN 151 −29.606 −36.120 39.598 1.000 12.36 ATOM 1128 CA GLN 151 −30.294 −36.665 40.759 1.000 19.45 ATOM 1129 CB GLN 151 −30.306 −35.680 41.932 1.000 12.11 ATOM 1130 CG GLN 151 −28.947 −35.446 42.561 1.000 16.34 ATOM 1131 CD GLN 151 −29.048 −34.481 43.734 1.000 22.05 ATOM 1132 OE1 GLN 151 −29.693 −34.803 44.729 1.000 39.76 ATOM 1133 NE2 GLN 151 −28.423 −33.317 43.598 1.000 16.49 ATOM 1134 C GLN 151 −31.745 −37.027 40.441 1.000 20.77 ATOM 1135 O GLN 151 −32.232 −38.044 40.936 1.000 19.36 ATOM 1136 N LEU 152 −32.397 −36.183 39.644 1.000 11.67 ATOM 1137 CA LEU 152 −33.818 −36.360 39.365 1.000 13.95 ATOM 1138 CB LEU 152 −34.438 −35.101 38.764 1.000 14.14 ATOM 1139 CG LEU 152 −34.837 −33.957 39.688 1.000 12.09 ATOM 1140 CD1 LEU 152 −34.781 −32.631 38.935 1.000 11.66 ATOM 1141 CD2 LEU 152 −36.225 −34.162 40.274 1.000 12.14 ATOM 1142 C LEU 152 −34.053 −37.544 38.428 1.000 13.07 ATOM 1143 O LEU 152 −34.913 −38.372 38.729 1.000 13.96 ATOM 1144 N ILE 153 −33.310 −37.613 37.326 1.000 13.21 ATOM 1145 CA ILE 153 −33.519 −38.661 36.334 1.000 12.12 ATOM 1146 CB ILE 153 −32.814 −38.377 34.991 1.000 9.74 ATOM 1147 CG2 ILE 153 −33.360 −37.106 34.355 1.000 0.00 ATOM 1148 CG1 ILE 153 −31.284 −38.333 35.061 1.000 8.16 ATOM 1149 CD1 ILE 153 −30.635 −38.332 33.684 1.000 0.00 ATOM 1150 C ILE 153 −33.054 −40.024 36.836 1.000 9.56 ATOM 1151 O ILE 153 −33.540 −41.043 36.342 1.000 4.79 ATOM 1152 N PHE 154 −32.138 −40.069 37.797 1.000 12.41 ATOM 1153 CA PHE 154 −31.645 −41.349 38.301 1.000 8.75 ATOM 1154 CB PHE 154 −30.113 −41.372 38.348 1.000 8.88 ATOM 1155 CG PHE 154 −29.456 −41.758 37.031 1.000 8.38 ATOM 1156 CD1 PHE 154 −28.597 −40.887 36.384 1.000 9.10 ATOM 1157 CD2 PHE 154 −29.703 −42.990 36.458 1.000 0.00 ATOM 1158 CE1 PHE 154 −28.000 −41.232 35.188 1.000 9.85 ATOM 1159 CE2 PHE 154 −29.119 −43.344 35.260 1.000 5.02 ATOM 1160 CZ PHE 154 −28.258 −42.468 34.624 1.000 8.39 ATOM 1161 C PHE 154 −32.199 −41.648 39.690 1.000 11.55 ATOM 1162 O PHE 154 −31.683 −42.515 40.400 1.000 10.77 ATOM 1163 N GLU 155 −33.246 −40.936 40.093 1.000 15.11 ATOM 1164 CA GLU 155 −33.898 −41.221 41.367 1.000 19.95 ATOM 1165 CB GLU 155 −35.134 −40.343 41.542 1.000 26.08 ATOM 1166 CG GLU 155 −35.558 −40.107 42.980 1.000 33.00 ATOM 1167 CD GLU 155 −36.339 −41.267 43.568 1.000 44.51 ATOM 1168 OE1 GLU 155 −37.432 −41.585 43.051 1.000 49.47 ATOM 1169 OE2 GLU 155 −35.862 −41.867 44.558 1.000 61.39 ATOM 1170 C GLU 155 −34.270 −42.702 41.449 1.000 18.82 ATOM 1171 O GLU 155 −34.978 −43.212 40.582 1.000 14.49 ATOM 1172 N GLY 156 −33.779 −43.376 42.481 1.000 12.58 ATOM 1173 CA GLY 156 −33.993 −44.787 42.696 1.000 6.50 ATOM 1174 C GLY 156 −33.061 −45.684 41.914 1.000 12.22 ATOM 1175 O GLY 156 −33.205 −46.914 41.914 1.000 27.90 ATOM 1176 N GLY 157 −32.082 −45.107 41.224 1.000 9.19 ATOM 1177 CA GLY 157 −31.216 −45.877 40.358 1.000 8.21 ATOM 1178 C GLY 157 −30.007 −46.514 40.991 1.000 8.61 ATOM 1179 O GLY 157 −29.563 −47.579 40.549 1.000 17.22 ATOM 1180 N GLU 158 −29.442 −45.887 42.018 1.000 7.58 ATOM 1181 CA GLU 158 −28.299 −46.453 42.721 1.000 7.50 ATOM 1182 CB GLU 158 −27.807 −45.505 43.814 1.000 9.84 ATOM 1183 CG GLU 158 −26.756 −46.097 44.739 1.000 11.00 ATOM 1184 CD GLU 158 −26.031 −45.053 45.564 1.000 24.40 ATOM 1185 OE1 GLU 158 −26.158 −43.845 45.267 1.000 33.57 ATOM 1186 OE2 GLU 158 −25.325 −45.439 46.523 1.000 39.11 ATOM 1187 C GLU 158 −28.696 −47.807 43.302 1.000 13.34 ATOM 1188 O GLU 158 −27.956 −48.787 43.225 1.000 29.78 ATOM 1189 N GLN 159 −29.895 −47.840 43.875 1.000 10.17 ATOM 1190 CA GLN 159 −30.481 −49.058 44.406 1.000 15.50 ATOM 1191 CB GLN 159 −31.856 −48.764 45.017 1.000 19.57 ATOM 1192 CG GLN 159 −32.548 −49.952 45.647 1.000 24.93 ATOM 1193 CD GLN 159 −31.737 −50.676 46.704 1.000 30.24 ATOM 1194 OE1 GLN 159 −31.940 −50.499 47.909 1.000 40.80 ATOM 1195 NE2 GLN 159 −30.800 −51.510 46.265 1.000 20.75 ATOM 1196 C GLN 159 −30.605 −50.132 43.336 1.000 17.89 ATOM 1197 O GLN 159 −30.218 −51.285 43.544 1.000 21.71 ATOM 1198 N LYS 160 −31.154 −49.791 42.168 1.000 15.99 ATOM 1199 CA LYS 160 −31.361 −50.855 41.176 1.000 6.75 ATOM 1200 CB LYS 160 −32.314 −50.369 40.090 1.000 10.24 ATOM 1201 CG LYS 160 −33.666 −49.907 40.607 1.000 6.13 ATOM 1202 CD LYS 160 −34.386 −49.041 39.581 1.000 11.21 ATOM 1203 CE LYS 160 −35.897 −49.190 39.702 1.000 9.55 ATOM 1204 NZ LYS 160 −36.616 −48.235 38.811 1.000 20.37 ATOM 1205 C LYS 160 −30.029 −51.305 40.591 1.000 14.32 ATOM 1206 O LYS 160 −29.842 −52.475 40.257 1.000 14.42 ATOM 1207 N THR 161 −29.082 −50.375 40.465 1.000 10.29 ATOM 1208 CA THR 161 −27.771 −50.734 39.933 1.000 13.43 ATOM 1209 CB THR 161 −26.878 −49.508 39.672 1.000 10.03 ATOM 1210 OG1 THR 161 −27.070 −48.557 40.730 1.000 30.01 ATOM 1211 CG2 THR 161 −27.263 −48.788 38.389 1.000 13.57 ATOM 1212 C THR 161 −27.057 −51.683 40.896 1.000 12.06 ATOM 1213 O THR 161 −26.160 −52.415 40.481 1.000 6.51 ATOM 1214 N THR 162 −27.457 −51.664 42.165 1.000 8.39 ATOM 1215 CA THR 162 −26.894 −52.551 43.177 1.000 9.75 ATOM 1216 CB THR 162 −27.286 −52.130 44.604 1.000 12.96 ATOM 1217 OG1 THR 162 −26.705 −50.863 44.941 1.000 11.98 ATOM 1218 CG2 THR 162 −26.735 −53.132 45.605 1.000 20.35 ATOM 1219 C THR 162 −27.349 −53.991 42.956 1.000 10.87 ATOM 1220 O THR 162 −26.764 −54.942 43.471 1.000 12.87 ATOM 1221 N GLU 163 −28.410 −54.170 42.174 1.000 16.58 ATOM 1222 CA GLU 163 −28.949 −55.496 41.905 1.000 20.69 ATOM 1223 CB GLU 163 −30.486 −55.450 41.861 1.000 21.36 ATOM 1224 CG GLU 163 −31.136 −54.918 43.122 1.000 19.81 ATOM 1225 CD GLU 163 −30.918 −55.799 44.332 1.000 20.57 ATOM 1226 OE1 GLU 163 −30.336 −56.894 44.181 1.000 13.38 ATOM 1227 OE2 GLU 163 −31.340 −55.394 45.441 1.000 37.36 ATOM 1228 C GLU 163 −28.455 −56.101 40.596 1.000 12.31 ATOM 1229 O GLU 163 −28.614 −57.306 40.384 1.000 8.17 ATOM 1230 N LEU 164 −27.880 −55.296 39.710 1.000 14.12 ATOM 1231 CA LEU 164 −27.561 −55.746 38.356 1.000 8.92 ATOM 1232 CB LEU 164 −26.960 −54.602 37.541 1.000 5.54 ATOM 1233 CG LEU 164 −27.903 −53.857 36.593 1.000 10.39 ATOM 1234 CD1 LEU 164 −29.295 −53.740 37.197 1.000 23.43 ATOM 1235 CD2 LEU 164 −27.352 −52.485 36.240 1.000 2.48 ATOM 1236 C LEU 164 −26.621 −56.943 38.361 1.000 6.54 ATOM 1237 O LEU 164 −26.847 −57.925 37.653 1.000 4.26 ATOM 1238 N ALA 165 −25.562 −56.865 39.159 1.000 7.24 ATOM 1239 CA ALA 165 −24.609 −57.965 39.239 1.000 11.41 ATOM 1240 CB ALA 165 −23.542 −57.659 40.276 1.000 11.40 ATOM 1241 C ALA 165 −25.312 −59.284 39.551 1.000 16.26 ATOM 1242 O ALA 165 −24.980 −60.302 38.947 1.000 18.13 ATOM 1243 N ARG 166 −26.266 −59.245 40.469 1.000 20.04 ATOM 1244 CA ARG 166 −27.014 −60.397 40.947 1.000 10.10 ATOM 1245 CB ARG 166 −27.875 −59.992 42.145 1.000 15.40 ATOM 1246 CG ARG 166 −28.600 −61.127 42.843 1.000 15.67 ATOM 1247 CD ARG 166 −29.286 −60.640 44.115 1.000 20.34 ATOM 1248 NE ARG 166 −30.097 −59.453 43.851 1.000 31.99 ATOM 1249 CZ ARG 166 −31.261 −59.505 43.202 1.000 37.46 ATOM 1250 NH1 ARG 166 −31.718 −60.673 42.770 1.000 41.26 ATOM 1251 NH2 ARG 166 −31.974 −58.410 42.979 1.000 44.85 ATOM 1252 C ARG 166 −27.899 −60.991 39.862 1.000 10.33 ATOM 1253 O ARG 166 −27.862 −62.186 39.569 1.000 11.28 ATOM 1254 N VAL 167 −28.724 −60.143 39.253 1.000 10.14 ATOM 1255 CA VAL 167 −29.647 −60.637 38.231 1.000 8.08 ATOM 1256 CB VAL 167 −30.800 −59.642 38.007 1.000 12.63 ATOM 1257 CG1 VAL 167 −31.873 −60.262 37.129 1.000 23.15 ATOM 1258 CG2 VAL 167 −31.423 −59.212 39.331 1.000 16.49 ATOM 1259 C VAL 167 −28.941 −60.943 36.916 1.000 8.93 ATOM 1260 O VAL 167 −29.342 −61.889 36.230 1.000 11.00 ATOM 1261 N TYR 168 −27.906 −60.209 36.507 1.000 6.53 ATOM 1262 CA TYR 168 −27.225 −60.549 35.262 1.000 5.82 ATOM 1263 CB TYR 168 −26.220 −59.494 34.815 1.000 12.35 ATOM 1264 CG TYR 168 −26.746 −58.249 34.148 1.000 10.53 ATOM 1265 CD1 TYR 168 −25.898 −57.415 33.429 1.000 4.25 ATOM 1266 CE1 TYR 168 −26.377 −56.273 32.816 1.000 3.59 ATOM 1267 CD2 TYR 168 −28.085 −57.889 34.230 1.000 9.22 ATOM 1268 CE2 TYR 168 −28.565 −56.750 33.624 1.000 11.67 ATOM 1269 CZ TYR 168 −27.708 −55.940 32.912 1.000 8.76 ATOM 1270 OH TYR 168 −28.194 −54.801 32.308 1.000 13.56 ATOM 1271 C TYR 168 −26.466 −61.863 35.444 1.000 9.45 ATOM 1272 O TYR 168 −26.398 −62.696 34.544 1.000 5.20 ATOM 1273 N SER 169 −25.896 −61.972 36.648 1.000 5.94 ATOM 1274 CA SER 169 −25.145 −63.174 36.999 1.000 11.65 ATOM 1275 CB SER 169 −24.663 −63.109 38.445 1.000 12.52 ATOM 1276 OG SER 169 −23.611 −64.024 38.688 1.000 13.86 ATOM 1277 C SER 169 −26.034 −64.389 36.740 1.000 14.93 ATOM 1278 O SER 169 −25.709 −65.240 35.912 1.000 25.35 ATOM 1279 N ALA 170 −27.161 −64.434 37.448 1.000 9.54 ATOM 1280 CA ALA 170 −28.154 −65.483 37.259 1.000 7.33 ATOM 1281 CB ALA 170 −29.397 −65.155 38.069 1.000 3.12 ATOM 1282 C ALA 170 −28.495 −65.659 35.785 1.000 12.27 ATOM 1283 O ALA 170 −28.526 −66.772 35.262 1.000 20.56 ATOM 1284 N LEU 171 −28.753 −64.558 35.081 1.000 15.11 ATOM 1285 CA LEU 171 −29.115 −64.661 33.665 1.000 17.04 ATOM 1286 CB LEU 171 −29.329 −63.272 33.076 1.000 13.64 ATOM 1287 CG LEU 171 −29.846 −63.164 31.645 1.000 21.08 ATOM 1288 CD1 LEU 171 −28.692 −63.043 30.658 1.000 45.18 ATOM 1289 CD2 LEU 171 −30.734 −64.340 31.270 1.000 17.34 ATOM 1290 C LEU 171 −28.052 −65.404 32.868 1.000 18.57 ATOM 1291 O LEU 171 −28.328 −66.409 32.219 1.000 17.64 ATOM 1292 N ALA 172 −26.825 −64.890 32.920 1.000 22.46 ATOM 1293 CA ALA 172 −25.735 −65.489 32.157 1.000 17.47 ATOM 1294 CB ALA 172 −24.454 −64.699 32.377 1.000 10.29 ATOM 1295 C ALA 172 −25.549 −66.953 32.536 1.000 13.15 ATOM 1296 O ALA 172 −25.192 −67.797 31.713 1.000 17.25 ATOM 1297 N SER 173 −25.802 −67.242 33.809 1.000 11.55 ATOM 1298 CA SER 173 −25.653 −68.595 34.337 1.000 15.80 ATOM 1299 CB SER 173 −25.837 −68.578 35.856 1.000 15.14 ATOM 1300 OG SER 173 −26.298 −69.837 36.293 1.000 15.66 ATOM 1301 C SER 173 −26.640 −69.565 33.691 1.000 10.39 ATOM 1302 O SER 173 −26.263 −70.667 33.284 1.000 5.06 ATOM 1303 N PHE 174 −27.882 −69.119 33.601 1.000 6.57 ATOM 1304 CA PHE 174 −28.970 −69.778 32.908 1.000 4.04 ATOM 1305 CB PHE 174 −30.288 −69.024 33.114 1.000 4.43 ATOM 1306 CG PHE 174 −31.524 −69.765 32.626 1.000 3.57 ATOM 1307 CD1 PHE 174 −32.219 −70.606 33.475 1.000 0.40 ATOM 1308 CD2 PHE 174 −31.988 −69.615 31.331 1.000 11.71 ATOM 1309 CE1 PHE 174 −33.343 −71.281 33.051 1.000 1.63 ATOM 1310 CE2 PHE 174 −33.114 −70.285 30.886 1.000 10.57 ATOM 1311 CZ PHE 174 −33.795 −71.119 31.756 1.000 10.59 ATOM 1312 C PHE 174 −28.701 −69.872 31.408 1.000 8.80 ATOM 1313 O PHE 174 −28.846 −70.949 30.834 1.000 0.14 ATOM 1314 N MET 175 −28.328 −68.751 30.793 1.000 7.91 ATOM 1315 CA MET 175 −28.058 −68.739 29.356 1.000 5.97 ATOM 1316 CB MET 175 −28.103 −67.321 28.780 1.000 0.00 ATOM 1317 CG MET 175 −29.492 −66.712 28.751 1.000 7.42 ATOM 1318 XD MET 175 −29.573 −65.056 28.023 1.000 16.37 ATOM 1319 CE MET 175 −30.064 −65.488 26.348 1.000 21.02 ATOM 1320 C MET 175 −26.715 −69.399 29.045 1.000 6.31 ATOM 1321 O MET 175 −26.332 −69.479 27.880 1.000 8.17 ATOM 1322 N LYS 176 −26.020 −69.872 30.070 1.000 8.77 ATOM 1323 CA LYS 176 −24.762 −70.598 29.939 1.000 10.68 ATOM 1324 CB LYS 176 −24.970 −71.945 29.239 1.000 10.45 ATOM 1325 CG LYS 176 −25.907 −72.900 29.971 1.000 3.74 ATOM 1326 CD LYS 176 −25.133 −73.755 30.964 1.000 5.05 ATOM 1327 CE LYS 176 −26.084 −74.568 31.833 1.000 6.09 ATOM 1328 NZ LYS 176 −26.739 −73.721 32.861 1.000 24.38 ATOM 1329 C LYS 176 −23.733 −69.760 29.190 1.000 12.34 ATOM 1330 O LYS 176 −23.084 −70.178 28.231 1.000 24.85 ATOM 1331 N VAL 177 −23.601 −68.520 29.648 1.000 12.09 ATOM 1332 CA VAL 177 −22.709 −67.581 28.953 1.000 12.10 ATOM 1333 CB VAL 177 −23.569 −66.629 28.106 1.000 9.74 ATOM 1334 CG1 VAL 177 −23.831 −65.319 28.835 1.000 18.59 ATOM 1335 CG2 VAL 177 −22.921 −66.372 26.753 1.000 20.30 ATOM 1336 C VAL 177 −21.848 −66.876 29.982 1.000 13.62 ATOM 1337 O VAL 177 −22.292 −66.730 31.126 1.000 20.25 ATOM 1338 N PRO 178 −20.635 −66.454 29.637 1.000 10.56 ATOM 1339 CD PRO 178 −20.019 −66.530 28.312 1.000 2.11 ATOM 1340 CA PRO 178 −19.760 −65.842 30.642 1.000 10.32 ATOM 1341 CB PRO 178 −18.433 −65.656 29.913 1.000 6.70 ATOM 1342 CG PRO 178 −18.623 −66.026 28.499 1.000 0.81 ATOM 1343 C PRO 178 −20.281 −64.483 31.119 1.000 20.65 ATOM 1344 O PRO 178 −20.796 −63.674 30.351 1.000 22.70 ATOM 1345 N PHE 179 −20.124 −64.253 32.412 1.000 22.55 ATOM 1346 CA PHE 179 −20.474 −63.025 33.107 1.000 19.13 ATOM 1347 CB PHE 179 −21.518 −63.283 34.194 1.000 8.91 ATOM 1348 CG PHE 179 −21.661 −62.215 35.268 1.000 8.12 ATOM 1349 CD1 PHE 179 −22.433 −61.087 35.044 1.000 10.36 ATOM 1350 CD2 PHE 179 −21.031 −62.337 36.499 1.000 2.04 ATOM 1351 CE1 PHE 179 −22.590 −60.103 36.004 1.000 2.43 ATOM 1352 CE2 PHE 179 −21.183 −61.367 37.470 1.000 0.76 ATOM 1353 CZ PHE 179 −21.963 −60.248 37.228 1.000 2.96 ATOM 1354 C PHE 179 −19.231 −62.400 33.736 1.000 13.74 ATOM 1355 O PHE 179 −18.309 −63.110 34.128 1.000 15.60 ATOM 1356 N PHE 180 −19.214 −61.080 33.838 1.000 14.28 ATOM 1357 CA PHE 180 −18.178 −60.371 34.573 1.000 13.03 ATOM 1358 CB PHE 180 −17.004 −59.952 33.686 1.000 17.94 ATOM 1359 CG PHE 180 −15.933 −59.164 34.433 1.000 21.76 ATOM 1360 CD1 PHE 180 −14.960 −59.807 35.176 1.000 21.38 ATOM 1361 CD2 PHE 180 −15.904 −57.780 34.391 1.000 19.62 ATOM 1362 CE1 PHE 180 −13.979 −59.108 35.859 1.000 15.07 ATOM 1363 CE2 PHE 180 −14.941 −57.064 35.075 1.000 21.73 ATOM 1364 CZ PHE 180 −13.979 −57.727 35.816 1.000 21.65 ATOM 1365 C PHE 180 −18.822 −59.164 35.256 1.000 12.16 ATOM 1366 O PHE 180 −19.594 −58.423 34.648 1.000 11.01 ATOM 1367 N ASP 181 −18.504 −58.988 36.536 1.000 7.72 ATOM 1368 CA ASP 181 −19.062 −57.864 37.286 1.000 10.61 ATOM 1369 CB ASP 181 −19.521 −58.346 38.659 1.000 5.77 ATOM 1370 CG ASP 181 −19.986 −57.225 39.559 1.000 4.11 ATOM 1371 OD1 ASP 181 −20.116 −56.076 39.092 1.000 8.61 ATOM 1372 OD2 ASP 181 −20.217 −57.508 40.750 1.000 11.49 ATOM 1373 C ASP 181 −18.037 −56.743 37.378 1.000 15.44 ATOM 1374 O ASP 181 −17.023 −56.872 38.060 1.000 16.84 ATOM 1375 N ALA 182 −18.293 −55.639 36.672 1.000 18.65 ATOM 1376 CA ALA 182 −17.359 −54.517 36.678 1.000 18.00 ATOM 1377 CB ALA 182 −17.778 −53.459 35.668 1.000 7.66 ATOM 1378 C ALA 182 −17.240 −53.911 38.075 1.000 18.92 ATOM 1379 O ALA 182 −16.198 −53.340 38.400 1.000 8.61 ATOM 1380 N GLY 183 −18.296 −54.044 38.872 1.000 15.67 ATOM 1381 CA GLY 183 −18.374 −53.516 40.219 1.000 13.53 ATOM 1382 C GLY 183 −17.444 −54.230 41.176 1.000 14.96 ATOM 1383 O GLY 183 −17.268 −53.846 42.330 1.000 25.31 ATOM 1384 N SER 184 −16.830 −55.306 40.696 1.000 16.38 ATOM 1385 CA SER 184 −15.940 −56.105 41.525 1.000 12.32 ATOM 1386 CB SER 184 −16.009 −57.574 41.116 1.000 14.55 ATOM 1387 OG SER 184 −15.237 −57.867 39.967 1.000 12.36 ATOM 1388 C SER 184 −14.516 −55.572 41.439 1.000 13.33 ATOM 1389 O SER 184 −13.644 −55.986 42.204 1.000 12.05 ATOM 1390 N VAL 185 −14.276 −54.640 40.515 1.000 9.89 ATOM 1391 CA VAL 185 −12.902 −54.156 40.358 1.000 14.54 ATOM 1392 CB VAL 185 −12.320 −54.649 39.021 1.000 16.34 ATOM 1393 CG1 VAL 185 −12.034 −56.141 39.100 1.000 13.09 ATOM 1394 CG2 VAL 185 −13.274 −54.346 37.877 1.000 20.34 ATOM 1395 C VAL 185 −12.802 −52.642 40.445 1.000 20.13 ATOM 1396 O VAL 185 −11.718 −52.101 40.682 1.000 11.67 ATOM 1397 N ILE 186 −13.912 −51.929 40.260 1.000 19.83 ATOM 1398 CA ILE 186 −13.905 −50.479 40.381 1.000 13.97 ATOM 1399 CB ILE 186 −13.716 −49.752 39.031 1.000 8.30 ATOM 1400 CG2 ILE 186 −12.362 −50.070 38.428 1.000 12.39 ATOM 1401 CG1 ILE 186 −14.830 −50.005 38.014 1.000 10.45 ATOM 1402 CD1 ILE 186 −14.956 −48.929 36.957 1.000 3.60 ATOM 1403 C ILE 186 −15.209 −49.957 40.979 1.000 13.38 ATOM 1404 O ILE 186 −16.256 −50.583 40.857 1.000 12.90 ATOM 1405 N SER 187 −15.120 −48.788 41.596 1.000 11.99 ATOM 1406 CA SER 187 −16.287 −48.046 42.052 1.000 9.16 ATOM 1407 CB SER 187 −16.110 −47.594 43.498 1.000 10.88 ATOM 1408 OG SER 187 −14.889 −46.879 43.658 1.000 16.58 ATOM 1409 C SER 187 −16.517 −46.839 41.145 1.000 11.87 ATOM 1410 O SER 187 −15.567 −46.304 40.563 1.000 16.73 ATOM 1411 N THR 188 −17.767 −46.410 41.015 1.000 15.17 ATOM 1412 CA THR 188 −18.077 −45.244 40.189 1.000 13.51 ATOM 1413 CB THR 188 −19.571 −45.151 39.848 1.000 12.88 ATOM 1414 OG1 THR 188 −19.969 −46.308 39.101 1.000 16.33 ATOM 1415 CG2 THR 188 −19.843 −43.943 38.961 1.000 8.08 ATOM 1416 C THR 188 −17.639 −43.978 40.916 1.000 14.09 ATOM 1417 O THR 188 −18.293 −43.535 41.860 1.000 10.72 ATOM 1418 N ASP 189 −16.518 −43.414 40.474 1.000 15.51 ATOM 1419 CA ASP 189 −15.911 −42.313 41.210 1.000 11.58 ATOM 1420 CB ASP 189 −14.407 −42.594 41.362 1.000 12.86 ATOM 1421 CG ASP 189 −14.158 −43.791 42.261 1.000 4.55 ATOM 1422 OD1 ASP 189 −14.915 −43.960 43.239 1.000 13.27 ATOM 1423 OD2 ASP 189 −13.208 −44.549 41.989 1.000 6.91 ATOM 1424 C ASP 189 −16.120 −40.949 40.567 1.000 15.34 ATOM 1425 O ASP 189 −15.910 −39.948 41.263 1.000 18.48 ATOM 1426 N GLY 190 −16.510 −40.918 39.303 1.000 19.39 ATOM 1427 CA GLY 190 −16.710 −39.718 38.515 1.000 15.08 ATOM 1428 C GLY 190 −17.385 −38.613 39.303 1.000 18.57 ATOM 1429 O GLY 190 −18.263 −38.908 40.119 1.000 20.64 ATOM 1430 N VAL 191 −16.952 −37.381 39.057 1.000 13.86 ATOM 1431 CA VAL 191 −17.428 −36.226 39.806 1.000 10.59 ATOM 1432 CB VAL 191 −16.825 −34.905 39.286 1.000 17.05 ATOM 1433 CG1 VAL 191 −15.324 −34.875 39.559 1.000 30.84 ATOM 1434 CG2 VAL 191 −17.092 −34.701 37.803 1.000 8.10 ATOM 1435 C VAL 191 −18.950 −36.129 39.774 1.000 10.47 ATOM 1436 O VAL 191 −19.542 −35.686 40.761 1.000 13.60 ATOM 1437 N ASP 192 −19.571 −36.534 38.668 1.000 1.46 ATOM 1438 CA ASP 192 −21.018 −36.447 38.540 1.000 0.70 ATOM 1439 CB ASP 192 −21.387 −36.356 37.056 1.000 2.10 ATOM 1440 CG ASP 192 −20.918 −37.566 36.268 1.000 9.82 ATOM 1441 OD1 ASP 192 −20.296 −38.478 36.857 1.000 8.20 ATOM 1442 OD2 ASP 192 −21.182 −37.597 35.047 1.000 6.78 ATOM 1443 C ASP 192 −21.754 −37.622 39.173 1.000 7.73 ATOM 1444 O ASP 192 −22.988 −37.674 39.136 1.000 7.10 ATOM 1445 N GLY 193 −21.027 −38.572 39.753 1.000 15.10 ATOM 1446 CA GLY 193 −21.631 −39.747 40.351 1.000 17.83 ATOM 1447 C GLY 193 −22.153 −40.758 39.352 1.000 18.93 ATOM 1448 O GLY 193 −22.820 −41.732 39.718 1.000 10.12 ATOM 1449 N ILE 194 −21.867 −40.565 38.062 1.000 11.77 ATOM 1450 CA ILE 194 −22.330 −41.546 37.081 1.000 7.87 ATOM 1451 CB ILE 194 −23.401 −40.945 36.154 1.000 9.95 ATOM 1452 CG2 ILE 194 −23.790 −41.927 35.063 1.000 0.00 ATOM 1453 CG1 ILE 194 −24.643 −40.441 36.896 1.000 9.90 ATOM 1454 CD1 ILE 194 −25.248 −39.237 36.206 1.000 8.85 ATOM 1455 C ILE 194 −21.191 −42.068 36.225 1.000 2.97 ATOM 1456 O ILE 194 −21.086 −43.251 35.924 1.000 6.72 ATOM 1457 N HIS 195 −20.277 −41.195 35.792 1.000 6.33 ATOM 1458 CA HIS 195 −19.256 −41.719 34.884 1.000 10.76 ATOM 1459 CB HIS 195 −19.089 −40.790 33.673 1.000 11.36 ATOM 1460 CG HIS 195 −20.402 −40.647 32.958 1.000 11.50 ATOM 1461 CD2 HIS 195 −20.981 −41.395 31.989 1.000 5.43 ATOM 1462 ND1 HIS 195 −21.283 −39.633 33.253 1.000 7.30 ATOM 1463 CE1 HIS 195 −22.351 −39.753 32.485 1.000 9.11 ATOM 1464 NE2 HIS 195 −22.192 −40.814 31.711 1.000 8.18 ATOM 1465 C HIS 195 −17.918 −41.941 35.577 1.000 8.63 ATOM 1466 O HIS 195 −17.762 −41.602 36.743 1.000 13.71 ATOM 1467 N PHE 196 −17.010 −42.529 34.812 1.000 6.37 ATOM 1468 CA PHE 196 −15.725 −43.017 35.249 1.000 9.06 ATOM 1469 CB PHE 196 −15.233 −44.136 34.320 1.000 5.38 ATOM 1470 CG PHE 196 −16.048 −45.412 34.451 1.000 10.20 ATOM 1471 CD1 PHE 196 −15.822 −46.481 33.602 1.000 8.01 ATOM 1472 CD2 PHE 196 −17.027 −45.509 35.427 1.000 6.21 ATOM 1473 CE1 PHE 196 −16.571 −47.637 33.722 1.000 11.17 ATOM 1474 CE2 PHE 196 −17.779 −46.662 35.546 1.000 14.06 ATOM 1475 CZ PHE 196 −17.549 −47.727 34.694 1.000 13.03 ATOM 1476 C PHE 196 −14.663 −41.925 35.273 1.000 12.92 ATOM 1477 O PHE 196 −14.757 −40.983 34.494 1.000 15.16 ATOM 1478 N THR 197 −13.694 −42.112 36.158 1.000 13.17 ATOM 1479 CA THR 197 −12.477 −41.318 36.183 1.000 17.95 ATOM 1480 CB THR 197 −11.886 −41.168 37.593 1.000 20.94 ATOM 1481 OG1 THR 197 −11.650 −42.458 38.173 1.000 20.14 ATOM 1482 CG2 THR 197 −12.882 −40.454 38.499 1.000 31.55 ATOM 1483 C THR 197 −11.443 −41.978 35.269 1.000 10.26 ATOM 1484 O THR 197 −11.713 −43.037 34.705 1.000 14.53 ATOM 1485 N GLU 198 −10.283 −41.362 35.133 1.000 9.05 ATOM 1486 CA GLU 198 −9.192 −41.943 34.362 1.000 12.89 ATOM 1487 CB GLU 198 −8.023 −40.960 34.314 1.000 20.40 ATOM 1488 CG GLU 198 −6.903 −41.349 33.362 1.000 32.30 ATOM 1489 CD GLU 198 −5.764 −40.346 33.328 1.000 35.77 ATOM 1490 OE1 GLU 198 −5.127 −40.141 34.385 1.000 42.59 ATOM 1491 OE2 GLU 198 −5.498 −39.761 32.256 1.000 25.40 ATOM 1492 C GLU 198 −8.779 −43.279 34.970 1.000 16.23 ATOM 1493 O GLU 198 −8.636 −44.296 34.292 1.000 14.85 ATOM 1494 N ALA 199 −8.596 −43.284 36.291 1.000 11.36 ATOM 1495 CA ALA 199 −8.233 −44.489 37.022 1.000 5.99 ATOM 1496 CB ALA 199 −8.047 −44.154 38.499 1.000 2.34 ATOM 1497 C ALA 199 −9.273 −45.594 36.873 1.000 7.89 ATOM 1498 O ALA 199 −8.922 −46.767 36.748 1.000 16.70 ATOM 1499 N ASN 200 −10.548 −45.210 36.897 1.000 13.48 ATOM 1500 CA ASN 200 −11.644 −46.155 36.715 1.000 11.59 ATOM 1501 CB ASN 200 −13.007 −45.474 36.805 1.000 4.12 ATOM 1502 CG ASN 200 −13.492 −45.192 38.209 1.000 11.67 ATOM 1503 OD1 ASN 200 −13.045 −45.767 39.200 1.000 6.19 ATOM 1504 ND2 ASN 200 −14.455 −44.276 38.330 1.000 13.74 ATOM 1505 C ASN 200 −11.505 −46.869 35.366 1.000 8.88 ATOM 1506 O ASN 200 −11.667 −48.084 35.305 1.000 9.08 ATOM 1507 N ASN 201 −11.208 −46.111 34.315 1.000 14.48 ATOM 1508 CA ASN 201 −11.074 −46.639 32.963 1.000 14.27 ATOM 1509 CB ASN 201 −10.903 −45.495 31.960 1.000 16.17 ATOM 1510 CG ASN 201 −12.221 −44.853 31.570 1.000 14.25 ATOM 1511 OD1 ASN 201 −13.050 −45.436 30.871 1.000 13.77 ATOM 1512 ND2 ASN 201 −12.441 −43.624 32.021 1.000 16.01 ATOM 1513 C ASN 201 −9.908 −47.620 32.870 1.000 12.95 ATOM 1514 O ASN 201 −10.050 −48.720 32.334 1.000 11.02 ATOM 1515 N ARG 202 −8.775 −47.207 33.412 1.000 15.80 ATOM 1516 CA ARG 202 −7.571 −48.020 33.532 1.000 14.85 ATOM 1517 CB ARG 202 −6.491 −47.250 34.294 1.000 17.85 ATOM 1518 CG ARG 202 −5.109 −47.874 34.325 1.000 17.66 ATOM 1519 CD ARG 202 −4.141 −47.026 35.143 1.000 19.69 ATOM 1520 NE ARG 202 −3.646 −45.881 34.388 1.000 30.64 ATOM 1521 CZ ARG 202 −2.410 −45.407 34.412 1.000 36.54 ATOM 1522 NH1 ARG 202 −1.470 −45.972 35.164 1.000 35.38 ATOM 1523 NH2 ARG 202 −2.093 −44.353 33.669 1.000 23.31 ATOM 1524 C ARG 202 −7.862 −49.344 34.229 1.000 6.52 ATOM 1525 O ARG 202 −7.636 −50.401 33.644 1.000 9.98 ATOM 1526 N ASP 203 −8.365 −49.285 35.464 1.000 3.83 ATOM 1527 CA ASP 203 −8.597 −50.500 36.237 1.000 12.72 ATOM 1528 CB ASP 203 −9.148 −50.181 37.631 1.000 9.96 ATOM 1529 CG ASP 203 −8.170 −49.370 38.458 1.000 16.04 ATOM 1530 OD1 ASP 203 −6.980 −49.324 38.086 1.000 18.66 ATOM 1531 OD2 ASP 203 −8.584 −48.772 39.474 1.000 22.09 ATOM 1532 C ASP 203 −9.548 −51.455 35.524 1.000 18.07 ATOM 1533 O ASP 203 −9.383 −52.674 35.579 1.000 12.38 ATOM 1534 N LEU 204 −10.550 −50.890 34.859 1.000 23.73 ATOM 1535 CA LEU 204 −11.541 −51.706 34.169 1.000 21.34 ATOM 1536 CB LEU 204 −12.745 −50.872 33.727 1.000 26.39 ATOM 1537 CG LEU 204 −14.123 −51.510 33.908 1.000 26.92 ATOM 1538 CD1 LEU 204 −15.079 −51.066 32.809 1.000 10.26 ATOM 1539 CD2 LEU 204 −14.019 −53.027 33.942 1.000 35.07 ATOM 1540 C LEU 204 −10.938 −52.392 32.948 1.000 10.84 ATOM 1541 O LEU 204 −11.212 −53.567 32.707 1.000 16.23 ATOM 1542 N GLY 205 −10.143 −51.649 32.189 1.000 8.26 ATOM 1543 CA GLY 205 −9.534 −52.173 30.984 1.000 6.27 ATOM 1544 C GLY 205 −8.472 −53.215 31.265 1.000 8.34 ATOM 1545 O GLY 205 −8.228 −54.094 30.436 1.000 9.21 ATOM 1546 N VAL 206 −7.829 −53.130 32.425 1.000 8.74 ATOM 1547 CA VAL 206 −6.833 −54.135 32.796 1.000 9.33 ATOM 1548 CB VAL 206 −5.942 −53.653 33.957 1.000 16.14 ATOM 1549 CG1 VAL 206 −5.020 −54.754 34.457 1.000 6.58 ATOM 1550 CG2 VAL 206 −5.124 −52.445 33.514 1.000 6.33 ATOM 1551 C VAL 206 −7.526 −55.447 33.154 1.000 5.34 ATOM 1552 O VAL 206 −7.118 −56.498 32.664 1.000 5.68 ATOM 1553 N ALA 207 −8.564 −55.384 33.982 1.000 4.56 ATOM 1554 CA ALA 207 −9.349 −56.547 34.369 1.000 8.39 ATOM 1555 CB ALA 207 −10.323 −56.180 35.490 1.000 0.79 ATOM 1556 C ALA 207 −10.144 −57.160 33.219 1.000 10.03 ATOM 1557 O ALA 207 −10.485 −58.346 33.261 1.000 13.69 ATOM 1558 N LEU 208 −10.471 −56.382 32.193 1.000 14.72 ATOM 1559 CA LEU 208 −11.278 −56.888 31.082 1.000 11.49 ATOM 1560 CB LEU 208 −12.065 −55.755 30.422 1.000 12.04 ATOM 1561 CG LEU 208 −13.325 −55.317 31.175 1.000 10.97 ATOM 1562 CD1 LEU 208 −13.985 −54.127 30.497 1.000 18.17 ATOM 1563 CD2 LEU 208 −14.302 −56.477 31.290 1.000 17.03 ATOM 1564 C LEU 208 −10.391 −57.604 30.067 1.000 6.10 ATOM 1565 O LEU 208 −10.857 −58.502 29.369 1.000 15.12 ATOM 1566 N ALA 209 −9.132 −57.191 30.019 1.000 10.78 ATOM 1567 CA ALA 209 −8.103 −57.815 29.203 1.000 16.00 ATOM 1568 CB ALA 209 −6.827 −56.992 29.220 1.000 18.55 ATOM 1569 C ALA 209 −7.829 −59.238 29.694 1.000 19.15 ATOM 1570 O ALA 209 −7.639 −60.143 28.882 1.000 13.89 ATOM 1571 N GLU 210 −7.822 −59.396 31.015 1.000 9.97 ATOM 1572 CA GLU 210 −7.645 −60.692 31.653 1.000 11.15 ATOM 1573 CB GLU 210 −7.535 −60.520 33.168 1.000 21.07 ATOM 1574 CG GLU 210 −6.097 −60.365 33.647 1.000 39.63 ATOM 1575 CD GLU 210 −5.696 −58.921 33.860 1.000 47.94 ATOM 1576 OE1 GLU 210 −5.958 −58.391 34.960 1.000 64.71 ATOM 1577 OE2 GLU 210 −5.097 −58.319 32.949 1.000 43.70 ATOM 1578 C GLU 210 −8.791 −61.634 31.308 1.000 10.80 ATOM 1579 O GLU 210 −8.589 −62.787 30.927 1.000 10.93 ATOM 1580 N GLN 211 −10.007 −61.120 31.441 1.000 10.29 ATOM 1581 CA GLN 211 −11.190 −61.871 31.035 1.000 17.12 ATOM 1582 CB GLN 211 −12.443 −61.052 31.363 1.000 15.73 ATOM 1583 CG GLN 211 −12.542 −60.709 32.844 1.000 19.97 ATOM 1584 CD GLN 211 −12.936 −61.923 33.671 1.000 20.12 ATOM 1585 OE1 GLN 211 −13.886 −62.628 33.331 1.000 17.44 ATOM 1586 NE2 GLN 211 −12.218 −62.166 34.759 1.000 12.84 ATOM 1587 C GLN 211 −11.146 −62.237 29.556 1.000 19.66 ATOM 1588 O GLN 211 −11.399 −63.384 29.170 1.000 12.73 ATOM 1589 N VAL 212 −10.822 −61.287 28.679 1.000 17.48 ATOM 1590 CA VAL 212 −10.785 −61.612 27.249 1.000 19.02 ATOM 1591 CB VAL 212 −10.426 −60.369 26.415 1.000 14.47 ATOM 1592 CG1 VAL 212 −10.189 −60.744 24.958 1.000 15.00 ATOM 1593 CG2 VAL 212 −11.527 −59.320 26.523 1.000 8.88 ATOM 1594 C VAL 212 −9.816 −62.745 26.936 1.000 23.29 ATOM 1595 O VAL 212 −10.192 −63.735 26.294 1.000 25.62 ATOM 1596 N ARG 213 −8.557 −62.645 27.361 1.000 21.16 ATOM 1597 CA ARG 213 −7.617 −63.740 27.126 1.000 22.08 ATOM 1598 CB ARG 213 −6.251 −63.462 27.752 1.000 19.45 ATOM 1599 CG ARG 213 −5.577 −62.178 27.300 1.000 20.41 ATOM 1600 CD ARG 213 −4.621 −61.690 28.380 1.000 26.40 ATOM 1601 NE ARG 213 −3.847 −60.527 27.952 1.000 29.86 ATOM 1602 CZ ARG 213 −3.556 −59.504 28.745 1.000 26.00 ATOM 1603 NH1 ARG 213 −3.968 −59.485 30.007 1.000 15.34 ATOM 1604 NH2 ARG 213 −2.847 −58.491 28.268 1.000 17.74 ATOM 1605 C ARG 213 −8.157 −65.052 27.695 1.000 21.76 ATOM 1606 O ARG 213 −7.893 −66.138 27.182 1.000 28.34 ATOM 1607 N SER 214 −8.924 −64.952 28.780 1.000 15.76 ATOM 1608 CA SER 214 −9.486 −66.151 29.389 1.000 15.09 ATOM 1609 CB SER 214 −10.043 −65.824 30.781 1.000 19.35 ATOM 1610 OG SER 214 −11.053 −66.745 31.144 1.000 46.77 ATOM 1611 C SER 214 −10.561 −66.790 28.529 1.000 15.48 ATOM 1612 O SER 214 −10.692 −68.016 28.535 1.000 24.87 ATOM 1613 N LEU 215 −11.355 −66.030 27.772 1.000 21.40 ATOM 1614 CA LEU 215 −12.367 −66.673 26.938 1.000 21.52 ATOM 1615 CB LEU 215 −13.655 −65.855 26.860 1.000 22.40 ATOM 1616 CG LEU 215 −14.176 −65.153 28.103 1.000 20.48 ATOM 1617 CD1 LEU 215 −15.071 −63.990 27.697 1.000 27.15 ATOM 1618 CD2 LEU 215 −14.931 −66.118 29.006 1.000 13.10 ATOM 1619 C LEU 215 −11.884 −66.920 25.510 1.000 20.60 ATOM 1620 O LEU 215 −12.536 −67.682 24.789 1.000 31.41 ATOM 1621 N LEU 216 −10.790 −66.303 25.077 1.000 21.43 ATOM 1622 CA LEU 216 −10.291 −66.503 23.718 1.000 19.55 ATOM 1623 CB LEU 216 −10.114 −65.148 23.021 1.000 19.47 ATOM 1624 CG LEU 216 −11.385 −64.305 22.870 1.000 16.11 ATOM 1625 CD1 LEU 216 −11.095 −63.042 22.076 1.000 17.60 ATOM 1626 CD2 LEU 216 −12.495 −65.108 22.211 1.000 4.00 ATOM 1627 C LEU 216 −8.983 −67.283 23.688 1.000 24.37 ATOM 1628 OT1 LEU 216 −8.472 −67.525 22.571 1.000 29.22 ATOM 1629 OT2 LEU 216 −8.463 −67.655 24.758 1.000 19.02

In addition to the above-described determinations, a carbamate-inhibited perhydrolase crystal was also produced and analyzed. In these experiments, a N-hexylcarbamate derivative of wild type perhydrolase was used. Wild-type perhydrolase (14.5 mg in 1 mL, 67 mM NaPO4 pH 7 buffer) was titrated at room temperature with 1.25 μL aliquots of 400 mM p-nitrophenyl-N-hexylcarbamate dissolved in DMSO. Perhydrolase activity was measured with p-nitrophenylbutyrate assay (See, Example 2), as a function of time after each addition of the inhibitor. Several additions over several hours were required for complete inhibition of the enzyme. After inhibition was complete, the buffer of the inhibited enzyme solution was exchanged for 10 mM HEPES pH 8.3. This solution was stored at −80° C. until used for crystallization screening experiments were conducted as described above. The inhibitor p-nitrophenyl-N-hexylcarbamate was prepared by methods known in the art (See e.g., Hosie et al., J. Biol. Chem., 262:260-264 [1987]). Briefly, the carbamate-inhibited perhydrolase was crystallized by vapor diffusion using the hanging drop method known in the art. A ml solution of inhibited perhydrolase (15 mgml in 10 mM HEPES, pH 8.2), was mixed with 4 μL of a reservoir solution (30% PEG-4,000 with 0.2 M lithium sulfate and 0.1 M Tris, pH 8.5) on a plastic coverslip, then inverted and sealed for a well of 6×4 Linbro plate containing 0.5 ml of the reservoir solution and allowed to equilibrate. Crystals formed within a few days. The crystals were flash frozen in liquid nitrogen and analyzed as described above.

While the native octamer was determined in space group P4 with unit cell dimensions:

a=98.184 b=98.184 and c=230.119 α=90.00 β=90.00 γ=90.00, this crystal diffracted to about 2.0 Å. The carbamate-inhibited crystal grew in the space group P1 with unit cell dimensions a=67.754, b=80.096, and c=85.974 α=104.10°, β=112.10°, and γ=97.40° and these crystals diffract to a resolution exceeding 1.0 Å.

The carbamate was bound in a manner to exploit the interactions between the keto oxygen of the carbamate and residues forming the oxyanion hole, the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobic side chain extends along the hydrophobic surface of the binding site out into the surface opening between pairs of dimers in the octamer structure. The carbamate moiety direction highlights the pivotal role of the S54V mutation. The hydrophobic moiety passes adjacent to the side chain of ser 54. Mutating the serine side to valine increased the hydrophobicity, and also served as a gatekeeper to prevent hydrophilic nucleophiles (e.g., water) for competing with desired deacylating nucleophiles. The t residues surrounding the carbamate moiety on the same and neighboring molecules forming the extended entry are expected to influence the selection of the optimal de-acylating nucleophile.

In addition, residues with surface-accessible side chain atoms were identified using the program “AreaMol,” within the CCP4 program package. Table 15-1 lists these residues. In this Table, the residue number, residue name, number of surface-accessible side chain atoms having at least 10.0 square atoms of accessible surface area, and maximum surface area (square angstroms) for any side chain atom within that residue (or CA for GLY residues) in the octameric structure of perhydrolase are provided.

TABLE 15-1 Surface-Accessible Side Chain Atoms Maximum Surface Residue Residue Number of Accessible Area (Square Number Name Side Chain Atoms Angstroms) 1 ALA 1 15.7 3 LYS′ 2 54.10 17 VAL 1 29.5 19 VAL 1 28.0 20 GLU 4 30.2 21 ASP 2 41.3 24 PRO 2 23.2 26 GLU 3 36.3 29 ALA 1 34.4 30 PRP 3 32.7 31 ASP 3 50.6 32 VAL 1 27.0 39 ALA 1 27.5 40 GLN 3 38.7 41 GLN 2 22.1 43 GLY 1 20.4 44 ALA 1 63.8 45 ASP 3 52.7 46 PHE 2 17.1 47 GLU 3 29.6 61 ASP 3 53.1 63 PRO 3 28.0 64 THR 1 15.7 65 ASP 1 10.8 66 PRO 3 33.5 67 ARG 2 20.3 69 ASN 1 11.0 72 SER 2 26.6 75 PRO 2 17.4 83 PRO 2 15.1 85 ASP 1 36.80 98 ALA 1 14.60 101 ARG 4 25.0 102 ARG 1 19.9 103 THR 1 43.7 104 PRO 1 17.90 105 LEU 1 10.1 113 VAL 1 17.3 116 THR 2 39.5 117 GLN 2 15.3 119 LEU 3 21.4 120 THR 2 34.1 122 ALA 1 38.0 123 GLY 1 11.0 126 GLY 1 11.9 128 THR 2 18.2 129 TYR 1 17.6 130 PRO 3 30.2 131 ALA 1 13.7 133 LYS 3 46.9 141 PRO 3 25.3 143 ALA 1 19.8 144 PRO 3 34.90 146 PRO 2 24.30 148 PRO 3 24.1 151 GLN 3 35.6 152 LEU 1 12.90 155 GLU 3 53.0 156 GLY 1 28.9 158 GLU 3 30.3 159 GLN 4 44.9 160 LYS 2 21.5 162 THR 2 25.0 163 GLU 2 23.3 165 ALA 1 23.1 169 SER 1 39.1 173 SER 2 33.3 174 PHE 1 11.1 175 MET 1 18.5 176 LYS 2 21.4 178 PRO 1 12.0 179 PHE 2 14.0 180 PHE 1 13.9 181 ASP 1 24.9 184 SER 1 27.0 185 VAL 1 27.5 187 SER 2 34.0 189 ASP 2 25.4 191 VAL 2 24.5 197 THR 2 21.6 198 GLU 3 43.5 199 ALA 1 50.5 202 ARG 3 37.2 203 ASP 2 30.9 206 VAL 2 45.2 210 GLU 3 34.6 211 GLN 2 19.6 213 ARG 5 30.8 214 SER 2 20.8 215 LEU 1 25.80

Example 16 Stain Removal

In this Example, experiments conducted to assess the stain removal abilities of perhydrolase are described.

Individual wells of 24 well culture plates were used to mimic conditions found in ordinary washing machines. Each well was filled with commercially available detergent (e.g., Ariel [Procter & Gamble], WOB [AATCC], and WFK [WFK]), and pre-stained cloth discs cut to fit inside of each well were added. Temperature and agitation were accomplished by attaching the plate to the inside of a common laboratory incubatorshaker. To measure bleaching effectiveness of the perhydrolase, fabric stained with tea (EMPA #167, available commercially from Test Fabrics) was used. A single cloth disc was placed in each well, and 1 ml of detergent liquid, containing enzyme, ester substrate, and peroxide was added. After agitation at 100-300 rpm @ 20-60° C., the fabric discs were removed, rinsed with tap water, and allowed to dry overnight. The reflectance of each individual cloth disc was measured, and plotted as an “L” value. These results are provided in FIG. 21, which shows that the addition of the perhydrolase of the present invention to the detergent consistently provides a greater degree of bleaching than the detergents alone. In this Figure, “E” indicates the results for each of the detergents tested in combination with the perhydrolase of the present invention.

Example 17 Cotton Bleaching

In this Example, experiments to assess the use of the perhydrolase of the present invention for bleaching of cotton fabrics are described.

In these experiments, six cotton swatches per canister were treated at 55° C. for 60 minutes in a Launder-O-meter. The substrates used in these experiments were: 3 (3″×3″) 428U and 3 (3″×3″) 400U per experiments. Two different types of 100% unbleached cotton fabrics from Testfabrics were tested (style 428U (desized but not bleached army carded cotton sateen); and style 400U (desized but not bleached cotton print cloth). The liquor ratio was about 26 to 1 (˜7.7 g fabric/˜200 ml volume liquor). The perhydrolase enzyme was tested at 12.7 mgP/ml, with ethyl acetate (3% (vv)), hydrogen peroxide (1500 ppm), and Triton X-100 (0.001%), in a sodium phosphate buffer (100 mM) for pH 7 and pH 8; as well as in a sodium carbonate (100 mM) buffer, for pH 9 and pH 10.

Bleaching effects were quantified with total color difference by taking 4 CIE L*a*b* values per each swatch before and after the treatments using a Chroma Meter CR-200 (Minolta), and total color difference of the swatches after the treatments were calculated according to the following:

Total color difference (ΔE)=√{square root over ((ΔL ² +Δa ² +Δb ²))}

(where ΔL, Δa, Δb, are differences in CIE L*, CIE a*, and CIE b* values respectively before and after the treatments).

Higher ΔE values indicate greater bleaching effects. The results (See, FIG. 22) indicated that the perhydrolase showed significantly improved bleaching effects on both types of 100% cotton fabrics at pH 7 and pH 8 under the conditions tested.

It was also observed that high amounts of motes (e.g., pigmented spots) disappeared on the enzyme treated substrates.

Example 18 Linen Bleaching

In this Example, experiments conducted to assess the linen bleaching capability of the perhydrolase of the present invention are described. The same methods and conditions as describe above for cotton testing (in Example 17) were used to test linen swatches. As indicated above, experiments were conduction in a Launder-O-meter using a linen fabric (linen suiting, Style L-53; Testfabrics).

In these experiments, 3 (4″×4″) linen swatches were treated with 12.7 mgP/ml of the perhydrolase enzyme with ethyl acetate (3% v/v), hydrogen peroxide (1200 ppm), and Triton X-100 (0.001%), in a sodium phosphate buffer (100 mM) for pH 7 and pH 8. The bleaching effects were calculated as described above in Example 17. FIG. 23 provides a graph showing the bleaching effects of the perhydrolase of the present invention tested at pH 7 and pH 8 on linen.

Example 19 Detergent Compositions

In the following Example, various detergent compositions are exemplified. In these formulations, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions. The abbreviated component identifications therein have the following meanings:

-   LAS: Sodium linear C₁₁₋₁₃ alkyl benzene sulfonate. -   TAS: Sodium tallow alkyl sulfate. -   CxyAS: Sodium C_(1x)-C_(1y), alkyl sulfate. -   CxyEz: C_(1x)-C_(1y) predominantly linear primary alcohol condensed     with an average of z moles of ethylene oxide. -   CxyAEzS: C_(1x)-C_(1y) sodium alkyl sulfate condensed with an     average of z moles of ethylene oxide. Added molecule name in the     examples. -   Nonionic: Mixed ethoxylatedpropoxylated fatty alcohol e.g. Plurafac     LF404 being an alcohol with an average degree of ethoxylation of 3.8     and an average degree of propoxylation of 4.5. -   QAS: R₂.N+(CH₃)₂(C₂H₄OH) with R₂=C₁₂-C₁₄. -   Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=1.6-3.2:1). -   Metasilicate: Sodium metasilicate (SiO₂:Na₂O ratio=1.0). -   Zeolite A: Hydrated Aluminosilicate of formula Na₁₂(AlO₂SiO₂)₁₂.     27H₂O -   SKS-6: Crystalline layered silicate of formula δ-Na₂Si₂O₅. -   Sulphate: Anhydrous sodium sulphate. -   STPP: Sodium Tripolyphosphate. -   MA/AA: Random copolymer of 4:1 acrylate/maleate, average molecular     weight about 70,000-80,000. -   AA: Sodium polyacrylate polymer of average molecular weight 4,500. -   Polycarboxylate: Copolymer comprising mixture of carboxylated     monomers such as acrylate, maleate and methyacrylate with a MW     ranging between 2,000-80,000 such as Sokolan commercially available     from BASF, being a copolymer of acrylic acid, MW4,500. -   BB1: 3-(3,4-Dihydroisoquinolinium)propane sulfonate -   BB2 1-(3,4-dihydroisoquinolinium)-decane-2-sulfate -   PB1: Sodium perborate monohydrate. -   PB4: Sodium perborate tetrahydrate of nominal formula NaBO₃.4H₂O. -   Percarbonate: Sodium percarbonate of nominal formula 2Na₂CO₃.3H₂O₂. -   TAED: Tetraacetyl ethylene diamine. -   NOBS: Nonanoyloxybenzene sulfonate in the form of the sodium salt. -   DTPA: Diethylene triamine pentaacetic acid. -   HEDP: 1,1-hydroxyethane diphosphonic acid. -   DETPMP: Diethyltriamine penta (methylene) phosphonate, marketed by     Monsanto under the Trade name Dequest 2060. -   EDDS: Ethylenediamine-N,N′-disuccinic acid, (S,S) isomer in the form     of its sodium salt -   Diamine: Dimethyl aminopropyl amine; 1,6-hezane diamine; 1,3-propane     diamine; 2-methyl-1,5-pentane diamine; 1,3-pentanediamine;     1-methyl-diaminopropane. -   DETBCHD 5,12-diethyl-1,5,8,12-tetraazabicyclo[6,6,2]hexadecane,     dichloride, Mn(II) salt -   PAAC: Pentaamine acetate cobalt(III) salt. -   Paraffin: Paraffin oil sold under the tradename Winog 70 by     Wintershall. -   Paraffin Sulfonate: A Paraffin oil or wax in which some of the     hydrogen atoms have been replaced by sulfonate groups. -   Aldose oxidase: Oxidase enzyme sold under the tradename Aldose     Oxidase by Novozymes A/S -   Galactose oxidase: Galactose oxidase from Sigma -   Protease: Proteolytic enzyme sold under the tradename Savinase,     Alcalase, Everlase by Novo Nordisk A/S, and the following from     Genencor International, Inc: “Protease A” described in U.S. Pat. No.     RE 34,606 in FIGS. 1A, 1B, and 7, and at column 11, lines 11-37;     “Protease B” described in U.S. Pat. No. 5,955,340 and U.S. Pat. No.     5,700,676 in FIGS. 1A, 1B and 5, as well as Table 1; and “Protease     C” described in U.S. Pat. No. 6,312,936 and U.S. Pat. No. 6,482,628     in FIGS. 1-3 [SEQ ID 3], and at column 25, line 12, “Protease D”     being the variant 101/G103A/104I/159/D232V/236H/245R/248D/252K (BPN′     numbering) described in WO 9920723. -   Amylase Amylolytic enzyme sold under the tradename Purafact Ox     Am^(R) described in WO 94/18314, WO96/05295 sold by Genencor;     Natalase®, Termamyl®, Fungamyl® and Duramyl®, all available from     Novozymes A/S. -   Lipase: Lipolytic enzyme sold under the tradename Lipolase Lipolase     Ultra by Novozymes A/S and Lipomax by Gist-Brocades. -   Cellulase: Cellulytic enzyme sold under the tradename Carezyme,     Celluzyme and/or Endolase by Novozymes A/S. -   Pectin Lyase: Pectaway® and Pectawash® available from Novozymes A/S. -   PVP: Polyvinylpyrrolidone with an average molecular weight of 60,000 -   PVNO: Polyvinylpyridine-N-Oxide, with an average molecular weight of     50,000. -   PVPVI: Copolymer of vinylimidazole and vinylpyrrolidone, with an     average molecular weight of 20,000. -   Brightener 1: Disodium 4,4′-bis(2-sulphostyryl)biphenyl. -   Silicone antifoam: Polydimethylsiloxane foam controller with     siloxane-oxyalkylene copolymer as dispersing agent with a ratio of     said foam controller to said dispersing agent of 10:1 to 100:1. -   Suds Suppressor: 12% Silicone/silica, 18% stearyl alcoho-1,70%     starch in granular form. -   SRP 1: Anionically end capped poly esters. -   PEG X: Polyethylene glycol, of a molecular weight of x. -   PVP K60®: Vinylpyrrolidone homopolymer (average MW 160,000) -   Jeffamine® ED-2001: Capped polyethylene glycol from Huntsman -   Isachem® AS: A branched alcohol alkyl sulphate from Enichem -   MME PEG (2000): Monomethyl ether polyethylene glycol (MW 2000) from     Fluka Chemie AG. -   DC3225C: Silicone suds suppresser, mixture of Silicone oil and     Silica from Dow Corning. -   TEPAE: Tetreaethylenepentaamine ethoxylate. -   BTA: Benzotriazole. -   Betaine: (CH₃)₃N⁺CH₂COO⁻ -   Sugar: Industry grade D-glucose or food grade sugar -   CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide -   TPKFA: C₁₂-C₁₄ topped whole cut fatty acids. -   Clay: A hydrated aluminumu silicate in a general formula     Al₂O₃SiO₂xH₂O. Types: Kaolinite, montmorillonite, atapulgite,     illite, bentonite, halloysite. -   MCAEM: Esters in the formula of R¹O_(x)[(R²)_(m)(R³)_(n)]_(p) -   pH: Measured as a 1% solution in distilled water at 20° C.

Example 20 Liquid Laundry Detergents

The following liquid laundry detergent compositions of the present invention are prepared.

I II III IV V LAS 18.0 — 6.0 — — C ₁₂-C₁₅ AE_(1.8)S — 2.0 8.0 11.0 5.0 C₈-C₁₀ propyl dimethyl 2.0 2.0 2.0 2.0 1.0 amine C₁₂-C₁₄ alkyl dimethyl — — — — 2.0 amine oxide C₁₂-C₁₅ AS — 17.0 — 7.0 8.0 CFAA — 5.0 4.0 4.0 3.0 C₁₂-C₁₄ Fatty alcohol 12.0 6.0 1.0 1.0 1.0 ethoxylate C₁₂-C₁₈ Fatty acid 11.0 11.0 4.0 4.0 3.0 Citric acid (anhydrous) 5.0 1.0 3.0 3.0 2.0 DETPMP 1.0 1.0 1.0 1.0 0.5 Monoethanolamine 11.0 8.0 5.0 5.0 2.0 Sodium hydroxide 1.0 1.0 2.5 1.0 1.5 Percarbonate — 3.5 — 2.5 — Propanediol 12.7 14.5 13.1 10. 8.0 Ethanol 1.8 1.8 4.7 5.4 1.0 Pectin Lyase — — — 0.005 — Amylase — 0.002 — — Cellulase — — 0.0002 0.0001 Lipase 0.1 — 0.1 — 0.1 Protease A 0.05 0.3 0.055 0.5 0.2 Aldose Oxidase 0.03 — 0.3 — 0.003 PAAC 0.01 0.01 — — — DETBCHD — — 0.02 0.01 — SRP1 0.5 0.5 — 0.3 0.3 Boric acid 2.4 2.4 2.8 2.8 2.4 Sodium xylene sulfonate — — 3.0 — — DC 3225C 1.0 1.0 1.0 1.0 1.0 2-butyl-octanol 0.03 0.04 0.04 0.03 0.03 DTPA 0.5 0.4 0.35 0.28 0.4 Brightener 1 0.18 0.10 0.11 — — Perhydrolase 0.05 0.3 0.08 0.5 0.2 MCAEM 3.0 8.0 12.0 1.5 4.8 (C₁₂-C₁₃ E _(6.5) Acetate) Balance to 100% perfume/dye and/or water

Example 21 Hand-Dish Liquid Detergent Compositions

The following hand dish liquid detergent compositions of the present invention are prepared.

I II III IV V VI C₁₂-C₁₅AE_(1.8)S 30.0 28.0 25.0 — 15.0 10.0 LAS — — — 5.0 15.0 12.0 Paraffin Sulfonate — — — 20.0 — — C₁₀-C₁₈ Alkyl 5.0 3.0 7.0 — — — Dimethyl Amine Oxide Betaine 3.0 — 1.0 3.0 1.0 — C₁₂ poly-OH fatty — — — 3.0 — 1.0 acid amide C₁₄ poly-OH fatty — 1.5 — — — — acid amide C₁₁E₉ 2.0 — 4.0 — — 20.0 DTPA — — — — 0.2 — Tri-sodium Citrate 0.25 — — 0.7 — — dihydrate Diamine 1.0 5.0 7.0 1.0 5.0 7.0 MgCl₂ 0.25 — — 1.0 — — Protease A 0.02 0.01 0.02 0.01 0.02 0.05 Amylase 0.001 — — 0.002 — 0.001 Aldose Oxidase 0.03 — 0.02 — 0.05 — Sodium Cumene — — — 2.0 1.5 3.0 Sulphonate PAAC 0.01 0.01 0.02 — — — DETBCHD — — — 0.01 0.02 0.01 PB1 1.5 2.8 1.2 — — — Perhydrolase 0.02 0.01 0.03 0.01 0.02 0.05 MCAEM 3.4 2.8 4.0 2.6 4.6 6.8 (C₁₄-C₁₅E₇ Acetate) Balance to 100% perfume/dye and/or water The pH of Compositions (I)-(VI) is about 8 to about 11

Example 22 Liquid Automatic Dishwashing Detergent

The following liquid automatic dishwashing detergent compositions of the present are prepared.

I II III IV V STPP 16 16 18 16 16 Potassium Sulfate — 10 8 — 10 1,2 propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid 4.0 3.0 3.0 4.0 3.0 CaCl₂ dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.5 0.5 0.5 0.5 0.5 Protease B 0.03 0.03 0.03 0.03 0.03 Amylase 0.02 — 0.02 0.02 — Aldose Oxidase — 0.15 0.02 — 0.01 Galactose Oxidase — — 0.01 — 0.01 PAAC 0.01 — — 0.01 — DETBCHD — 0.01 — — 0.01 Perhydrolase 0.1 0.03 0.05 0.03 0.06 MCAEM 5.0 3.0 12.0 8.0 1.0 (C₁₄-C₁₅E₁₂ Acetate) Balance to 100% perfume/dye and/or water

Example 23 Laundry Compositions

The following laundry compositions of present invention, which may be in the form of granules or tablet, are prepared.

Base Product I II III IV V C₁₄-C₁₅AS or TAS 8.0 5.0 3.0 3.0 3.0 LAS 8.0 — 8.0 — 7.0 C₁₂-C₁₅AE₃S 0.5 2.0 1.0 — — C₁₂-C₁₅E₅ or E₃ 2.0 — 5.0 2.0 2.0 QAS — — — 1.0 1.0 Zeolite A 20.0 18.0 11.0 — 10.0 SKS-6 (dry add) — — 9.0 — — MA/AA 2.0 2.0 2.0 — — AA — — — — 4.0 3Na Citrate 2H₂O — 2.0 — — — Citric Acid (Anhydrous) 2.0 — 1.5 2.0 — DTPA 0.2 0.2 — — — EDDS — — 0.5 0.1 — HEDP — — 0.2 0.1 — PB1 3.0 4.8 — — 4.0 Percarbonate — — 3.8 5.2 — NOBS 1.9 — — — — NACA OBS — — 2.0 — — TAED 0.5 2.0 2.0 5.0 1.00 BB1 0.06 — 0.34 — 0.14 BB2 — 0.14 — 0.20 — Anhydrous Na Carbonate 15.0 18.0 8.0 15.0 15.0 Sulfate 5.0 12.0 2.0 17.0 3.0 Silicate — 1.0 — — 8.0 Protease B 0.033 0.033 — — — Protease C — — 0.033 0.046 0.033 Lipase — 0.008 — — — Amylase 0.001 — — — 0.001 Cellulase — 0.0014 — — — Pectin Lyase 0.001 0.001 0.001 0.001 0.001 Aldose Oxidase 0.03 — 0.05 — — PAAC — 0.01 — — 0.05 Perhydrolase 0.03 0.05 1.0 0.06 0.1 MCAEM** 2.0 5.0 12.0 3.5 6.8 Balance to 100% Moisture and/or Minors* Perfume/Dye, Brightener/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/High Molecular PEG/Clay. **MCAEM is selected from the group consisting of C ₉-C₁₁E_(2.5) Acetate, [C₁₂H₂₅N(CH₃)(CH₂CH₂OAc)₂]⁺ Cl⁻, (CH₃)₂NCH₂CH₂OCH₂CH₂OAc, or mixtures thereof.. Balance to 100% Moisture and/or Minors*

-   -   Perfume/Dye, Brightener/SRP1/Na         Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds         suppressor/High Molecular PEG/Clay.     -   ** MCAEM is selected from the group consisting of C₉-C₁₁E_(2.5)         Acetate, [C₁₂H₂₅N(CH₃)(CH₂CH₂OAc)₂]⁺Cl⁻,         (CH₃)₂NCH₂CH₂OCH₂CH₂OAc, or mixtures thereof . . . .

Example 24 Liquid Laundry Detergents

The following liquid laundry detergent formulations of the present invention are prepared.

I I II III IV V LAS 11.5 11.5 9.0 — 4.0 — C₁₂-C₁₅AE_(2.85)S — — 3.0 18.0 — 16.0 C₁₄-C₁₅E_(2.5)S 11.5 11.5 3.0 — 16.0 — C₁₂-C₁₃E₉ — — 3.0 2.0 2.0 1.0 C₁₂-C₁₃E₇ 3.2 3.2 — — — — CFAA — — — 5.0 — 3.0 TPKFA 2.0 2.0 — 2.0 0.5 2.0 Citric Acid 3.2 3.2 0.5 1.2 2.0 1.2 (Anhydrous) Ca formate 0.1 0.1 0.06 0.1 — — Na formate 0.5 0.5 0.06 0.1 0.05 0.05 Na Culmene 4.0 4.0 1.0 3.0 1.2 — Sulfonate Borate 0.6 0.6 — 3.0 2.0 3.0 Na hydroxide 6.0 6.0 2.0 3.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1,2 Propanediol 3.0 3.0 2.0 8.0 8.0 5.0 Mono- 3.0 3.0 1.5 1.0 2.5 1.0 ethanolamine TEPAE 2.0 2.0 — 1.0 1.0 1.0 PB1 — 4.5 — 2.8 — Protease A 0.03 0.03 0.01 0.03 0.02 0.02 Lipase — — — 0.002 — — Amylase — — — — 0.002 — Cellulase — — — — — 0.0001 Pectin Lyase 0.005 0.005 — — — Aldose Oxidase 0.05 — — 0.05 — 0.02 Galactose — 0.04 oxidase Perhydrolase 0.03 0.05 0.01 0.03 0.08 0.02 MCAEM 3.2 4.6 1.8 3.5 6.2 2.8 (C₁₂-C₁₂E₆ Acetate) PAAC 0.03 0.03 0.02 — — — DETBCHD — — — 0.02 0.01 — SRP 1 0.2 0.2 — 0.1 — — DTPA — — — 0.3 — — PVNO — — — 0.3 — 0.2 Brightener 1 0.2 0.2 0.07 0.1 — — Silicone 0.04 0.04 0.02 0.1 0.1 0.1 antifoam Balance to 100% perfume/dye, and/or water Balance to 100% perfume dye, and/or water

Example 25 Compact High-Density Dishwashing Detergents

The following compact high density dishwashing detergent of the present invention are prepared:

I II III IV V VI STPP — 45.0 45.0 — — 40.0 3Na Citrate 2H₂O 17.0 — — 50.0 40.2 — Na Carbonate 17.5 14.0 20.0 — 8.0 33.6 Bicarbonate — — — 26.0 — — Silicate 15.0 15.0 8.0 — 25.0 3.6 Metasilicate 2.5 4.5 4.5 — — — PB1 — — 4.5 — — — PB4 — — — 5.0 — — Percarbonate — — — — — 4.8 BB1 — 0.1 0.1 — 0.5 — BB2 0.2 0.05 — 0.1 — 0.6 Nonionic 2.0 1.5 1.5 3.0 1.9 5.9 HEDP 1.0 — — — — — DETPMP 0.6 — — — — — PAAC 0.03 0.05 0.02 — — — Paraffin 0.5 0.4 0.4 0.6 — — Protease B 0.072 0.053 0.053 0.026 0.059 0.01 Amylase 0.012 — 0.012 — 0.021 0.006 Lipase — 0.001 — 0.005 — — Pectin Lyase 0.001 0.001 0.001 — — — Aldose Oxidase 0.05 0.05 0.03 0.01 0.02 0.01 Perhydrolase 0.072 0.053 0.053 0.026 0.059 0.01 MCAEM 3.5 2.8 1.6 7.5 4.2 0.8 (C₁₂-C₁₃E_(6.5) Acetate) BTA 0.3 0.2 0.2 0.3 0.3 0.3 Polycarboxylate 6.0 — — — 4.0 0.9 Perfume 0.2 0.1 0.1 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/High Molecular PEG/Clay. The pH of compositions (I) through (VI) is from about 9.6 to about 11.3. Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/High Molecular PEG/Clay. The pH of compositions (I) through (VI) is from about 9.6 to about 11.3.

Example 26 Tablet Detergent Compositions

The following tablet detergent compositions of the present invention are prepared by compression of a granular dishwashing detergent composition at a pressure of 13KNcm² using a standard 12 head rotary press.

I II III IV V VI VII VIII STPP — 48.8 44.7 38.2 — 42.4 46.1 36.0 3Na Citrate 2H₂O 20.0 — — — 35.9 — — — Na Carbonate 20.0 5.0 14.0 15.4 8.0 23.0 20.0 28.0 Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.3 4.2 Lipase 0.001 — 0.01 — 0.02 — — — Protease B 0.042 0.072 0.042 0.031 — — — — Protease C — — — — 0.052 0.023 0.023 0.029 Perhydrolase 0.01 0.08 0.05 0.04 0.052 0.023 0.023 0.029 MCAEM 2.8 6.5 4.5 3.8 4.6 2.8 2.8 2.8 (C₁₂-C₁₃E_(6.5) Acetate) Amylase 0.012 0.012 0.012 — 0.015 — 0.017 0.002 Pectin Lyase 0.005 — — 0.002 — — — — Aldose Oxidase — 0.03 — 0.02 0.02 — 0.03 — PB1 — — 3.8 — 7.8 — — 8.5 Percarbonate 6.0 — — 6.0 — 5.0 — — BB1 0.2 — 0.5 — 0.3 0.2 — — BB2 — 0.2 — 0.5 — — 0.1 0.2 Nonionic 1.5 2.0 2.0 2.2 1.0 4.2 4.0 6.5 PAAC 0.01 0.01 0.02 — — — — — DETBCHD — — — 0.02 0.02 — — — TAED — — — — — 2.1 — 1.6 HEDP 1.0 — — 0.9 — 0.4 0.2 — DETPMP 0.7 — — — — — — — Paraffin 0.4 0.5 0.5 0.5 — — 0.5 — BTA 0.2 0.3 0.3 0.3 0.3 0.3 0.3 — Polycarboxylate 4.0 — — — 4.9 0.6 0.8 — PEG 400-30,000 — — — — — 2.0 — 2.0 Glycerol — — — — — 0.4 — 0.5 Perfume — — — 0.05 0.2 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/High Molecular PEG/Clay. The pH of Compositions (I) through 7(VIII) is from about 10 to about 11.5. The tablet weight of Compositions 7(I) through 7(VIII) is from about 20 grams to about 30 grams. Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/High Molecular PEG/Clay. The pH of Compositions (I) through 7(VIII) is from about 10 to about 11.5. The tablet weight of Compositions 7(I) through 7(VIII) is from about 20 grams to about 30 grams.

Example 27 Liquid Hard Surface Cleaning Detergents

The following liquid hard surface cleaning detergent compositions of the present invention are prepared.

I II III IV V VI VII C₉-C₁₁E₅ 2.4 1.9 2.5 2.5 2.5 2.4 2.5 C₁₂-C₁₄E₅ 3.6 2.9 2.5 2.5 2.5 3.6 2.5 C₇-C₉E₆ — — — — 8.0 — — C₁₂-C₁₄E₂₁ 1.0 0.8 4.0 2.0 2.0 1.0 2.0 LAS — — — 0.8 0.8 — 0.8 Sodium culmene sulfonate 1.5 2.6 — 1.5 1.5 1.5 1.5 Isachem ® AS 0.6 0.6 — — — 0.6 — Na₂CO₃ 0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na Citrate 2H₂O 0.5 0.56 0.5 0.6 0.75 0.5 0.75 NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.13 0.6 0.1 0.4 0.6 0.4 2-butyl octanol 0.3 0.3 — 0.3 0.3 0.3 0.3 PEG DME-2000 ® 0.4 — 0.3 0.35 0.5 — — PVP 0.3 0.4 0.6 0.3 0.5 — — MME PEG (2000) ® — — — — — 0.5 0.5 Jeffamine ® ED-2001 — 0.4 — — 0.5 — — PAAC — — — 0.03 0.03 0.03 — DETBCHD 0.03 0.05 0.05 — — — — Protease B 0.07 0.05 0.05 0.03 0.06 0.01 0.04 Amylase 0.12 0.01 0.01 — 0.02 — 0.01 Lipase — 0.001 — 0.005 — 0.005 — Perhydrolase 0.07 0.05 0.08 0.03 0.06 0.01 0.04 MCAEM (C₁₂-C₁₅E₈ 3.5 5.6 4.8 5.3 3.6 8.0 4.7 Acetate) Pectin Lyase 0.001 — 0.001 — — — 0.002 PB1 — 4.6 — 3.8 — — — Aldose Oxidase 0.05 — 0.03 — 0.02 0.02 0.05 Balance to 100% perfume/dye, and/or water The pH of Compositions (I) through (VII) is from about 7.4 to about 9.5. Balance to 100% perfume dye, and/or water The pH of Compositions (I) through (VII) is from about 7.4 to about 9.5.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.

Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. 

1-166. (canceled)
 167. A cleaning, bleaching or disinfecting composition, comprising an isolated perhydrolase, which is a homolog of M. smegmatis perhydrolase and is a SGNH-hydrolase family polypeptide, which comprises: a) the motifs GDSL-GRTT, and GAND; b) the amino acid residues corresponding to L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, 180, G205, S11, D192, and H195 of SEQ ID NO: 2; wherein said perhydrolase exhibits: i) perhydrolysis activity that is at least about 0.2 compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase; and ii) a perhydrolysis to hydrolysis ratio that is greater than 1; wherein said perhydrolysis activity and said perhydrolysis to hydrolysis ratio is determined using the perhydrolysis and hydrolysis assays as described in Example
 2. 168. The composition of claim 167, wherein said perhydrolase has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:
 97. 169. The composition of claim 168, wherein said perhydrolase is Mesorhizobium loti Mlo I (Q98MY5) and comprises the amino acid sequence as set out in SEQ ID NO:
 97. 170. A cleaning composition according to any one of claims 167, comprising: a) at least 0.0001 weight percent of said perhydrolase; b) a molecule comprising an ester moiety; and c) optionally, an adjunct ingredient.
 171. The cleaning composition of claim 170, wherein said composition comprises: a) at least 0.0001 weight percent of said perhydrolase; b) a material selected from the group consisting of: a peroxygen source, hydrogen peroxide and mixtures thereof, said peroxygen source being selected from the group consisting of: i) a per-salt; ii) an organic peroxyacid; iii) urea hydrogen peroxide; iv) a carbohydrate and carbohydrate oxidase mixture, and v) mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient.
 172. The cleaning composition of claim 170, wherein said adjunct ingredient is selected from the group consisting of: surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removalanti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof.
 173. The cleaning composition of claim 171, wherein: a) said per-salt is selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof; b) said carbohydrate is selected from the group consisting of mono-carbohydrates, di-carbohydrates, tri-carbohydrates, oligo-carbohydrates and mixtures thereof; c) said carbohydrate oxidase is selected from the group consisting of aldose oxidase (IUPAC classification EC1.1.3.9), galactose oxidase (IUPAC classification EC1.1.3.9), cellobiose oxidase (IUPAC classification EC1.1.3.25), pyranose oxidase (IUPAC classification EC1.1.3.10), sorbose oxidase (IUPAC classification EC1.1.3.11) hexose oxidase (IUPAC classification EC1.1.3.5). glucose oxidase (IUPAC classification EC1.1.3.4) and mixtures thereof; and d) said molecule comprising an ester moiety has the formula: R¹O_(x)[(R²)_(m)(R³)_(n)]_(p) i) wherein R¹ is a moiety selected from the group consisting of H, substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; ii) each R² is an alkoxylate moiety; iii) R³ is an ester-forming moiety having the formula: R⁴CO— wherein R⁴ is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; iv) x is 1 when R¹ is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R¹; v) p is an integer that is equal to or less than x; vi) m is an integer from 0 to 50; and vii) n is at least
 1. 174. The cleaning composition of claim 173, wherein: a) R¹ is an C₂-C₃₂ substituted or unsubstituted alkyl or heteroalkyl moiety; b) each R² is independently an ethoxylate or propoxylate moiety; c) m is an integer from 1 to 12; and d) R³ is an ester-forming moiety having the formula: R⁴CO— wherein R⁴ is: i) a substituted or unsubstituted alkyl, alkenyl or alkynyl moiety comprising from 1 to 22 carbon atoms; or ii) a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety comprising from 4 to 22 carbon atoms.
 175. The cleaning composition of claim 170, wherein the molecule comprising the ester moiety has the formula: R¹O_(x)[(R²)_(m)(R³)_(n)]_(p) wherein: a) R¹ is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, said R¹ moiety that comprises an amine moiety being selected from the group consisting of substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; b) each R² is an alkoxylate moiety; c) R³ is an ester-forming moiety having the formula: R⁴CO— wherein R⁴ may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; d) x is 1 when R¹ is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R¹; e) p is an integer that is equal to or less than x f) m is an integer from 0 to 12; and g) n is at least 1; and optionally wherein said molecule comprising an ester moiety has a weight average molecular weight of less than 600,000 Daltons.
 176. The cleaning composition of claim 170, wherein said molecule comprising an ester moiety is selected from triacetin, ethylene glycol diacetate and propylene glycol diacetate.
 177. The composition of claim 167, wherein said composition is a bleaching or disinfecting composition, further comprising at least one additional enzyme or enzyme derivative selected from the group consisting of: proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.
 178. A method of disinfecting and/or bleaching a surface or an article, the method comprising contacting said surface or article with the composition of claim 167, optionally wherein the surface or article is selected from textiles, hard surfaces, paper, pulp, hair, teeth, skin, medical devices, medical equipment, industrial equipment and fermenters.
 179. A method of cleaning comprising the steps of: a) contacting a surface and/or an article comprising a fabric with a cleaning composition of claim 167; and b) optionally washing and/or rinsing the surface or material. 